Pharmaceuticals for Oral Delivery

ABSTRACT

The present invention provides pharmaceutical compositions suitable for oral delivery and methods of treating subjects in need thereof. The pharmaceutical compositions of the present invention enhance bioavailability of at least one compound classified as BCS Class II, BCS Class III or BCS Class IV.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/353,213, filed Nov. 16, 2016, which is a continuation of U.S.application Ser. No. 15/184,971, filed on Jun. 16, 2016, now U.S. Pat.No. 9,526,785, which is a continuation of U.S. application Ser. No.14/197,405, filed on Mar. 5, 2014, now U.S. Pat. No. 9,457,086, whichclaims priority to and the benefit of U.S. Provisional Application No.61/772,927, filed Mar. 5, 2013 and U.S. Provisional Application No.61/925,443, filed Jan. 9, 2014. The contents of each of theseapplications are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

The biopharmaceutical classification system (“BCS”) guidance waspublished in 2000 by the U.S. Food and Drug Administration (“FDA”), tostandardize oral formulation development that currently forms the basisof the scientific framework used for classifying drug substances basedon their aqueous solubility and intestinal permeability. According tothe BCS, drug substances are classified into four classes based solelyon their solubility and intestinal permeability: Class I: HighSolubility, High Permeability; Class II: Low Solubility, HighPermeability; Class III: High Solubility, Low Permeability and Class IV:Low Solubility, Low Permeability. Poor solubility leads to significanthurdles in the oral absorption and bioavailability of the drug candidateby decreasing its dissolution rate and membrane permeation. Permeabilityacross biological membranes is a key factor in the absorption anddistribution of drugs. Poor permeability can lead to poor absorptionacross the gastrointestinal mucosa or poor distribution throughout thebody.

Poor oral bioavailability (“F”) is one of the leading causes of compoundfailure in preclinical and clinical development. Compounds with poororal F tend to have low plasma exposure and high interindividualvariability, which limits their therapeutic usefulness. Thus, there is aneed in the art for pharmaceutical compositions that improve oralbioavailability. The present invention addresses these needs.

SUMMARY OF THE INVENTION

The present invention provides a pharmaceutical composition suitable fororal delivery including at least one compound classified as BCS ClassII, BCS Class III or BCS Class IV; at least one absorption enhancer; atleast one pH lowering compound; and at least one chelating agent.

The present invention also provides methods for enhancing thebioavailability of a therapeutically effective amount of at least onecompound classified as BCS Class II, BCS Class III or BCS Class IVincluding orally administering a pharmaceutical composition including atleast one compound classified as BCS Class II, BCS Class III or BCSClass IV; at least one absorption enhancer; at least one pH loweringcompound; and at least one chelating agent.

The present invention also provides methods of treating a bacterial orviral infection in a subject in need thereof including orallyadministering a pharmaceutical composition including at least onecompound classified as BCS Class II, BCS Class III or BCS Class IV; atleast one absorption enhancer; at least one pH lowering compound; and atleast one chelating agent. The bacterial infection can be agram-positive or gram-negative infection.

The present invention also provides methods of treating complicated skinand skin structure infections (cSSSI) in a subject in need thereofincluding orally administering a pharmaceutical composition including atleast one compound classified as BCS Class II, BCS Class III or BCSClass IV; at least one absorption enhancer; at least one pH loweringcompound; and at least one chelating agent.

The at least one compound classified as BCS Class II, BCS Class III orBCS Class IV can be a small molecule organic compound. The at least onecompound classified as BCS Class II, BCS Class III or BCS Class IV canbe an antibiotic or antiviral compound. The at least one compoundclassified as BCS Class II, BCS Class III or BCS Class IV can be atigecycline, zanamivir, kanamycin, tobramycin or fenofibrate.

The pharmaceutical composition can be a solid dosage pharmaceuticalcomposition, the pharmaceutical composition can be a multilayer soliddosage pharmaceutical composition.

The pH lowering compound can have a pKa no higher than 4.2, the pHlowering compound can have a pKa no higher than 3.0. Preferably, the pHlowering agent is sodium citrate.

The chelating agent can be a carboxylic acid chelating agent or an aminoacid chelating agent. The carboxylic acid chelating agent can beacetylsalicylic acid, acetic acid, ascorbic acid, citric acid, fumaricacid, glucuronic acid, glutaric acid, glyceric acid, glycocolic acid,glyoxic acid, isocitric acid, isovaleric acid, lactic acid, maleic acid,oxaloacetic acid, oxalosuccinic acid, propionic acid, pyruvic acid,succinic acid, tartaric acid, or valeric acid. Preferably, thecarboxylic acid chelating agent is citric acid.

The at least one absorption enhancer can include an acylcarnitine.Preferably, the acylcarnitine is lauroyl carnitine. The at least oneabsorption enhancer can include a surface acting agent. The surfaceacting agent can be an acid soluble bile acid.

The pharmaceutical composition can further include a cationic surfaceacting agent.

The pharmaceutical composition can further include an acid resistantprotective vehicle. Preferably, the acid resistant protective vehicle isa viscous protective syrup.

A multilayered solid dosage pharmaceutical composition including achelating agent and an acid resistant protective vehicle can alsoinclude a water soluble barrier is layered between the chelating agentand the acid resistant protective vehicle.

The present invention provides a pharmaceutical composition suitable fororal delivery including: at least one antibiotic or antiviral compoundclassified as BCS Class II, BCS Class III or BCS Class IV;lauroyl-carnitine; citric acid; and sodium citrate, wherein thecomposition is buffered at pH 3.5.

While the disclosure has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the disclosure, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

While this disclosure has been particularly shown and described withreferences to preferred aspects thereof, it will be understood by thoseskilled in the art that various changes in form and details may be madetherein without departing from the scope of the disclosure encompassedby the appended claims.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. Genbank and NCBI submissions indicated byaccession number cited herein are hereby incorporated by reference. Allother published references, documents, manuscripts and scientificliterature cited herein are hereby incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing Individual and Mean (±SD) Plasma TigecyclineConcentration in Sprague-Dawley Rats Following a Single Dose IV BolusInjection of 0.64 mg/kg.

FIG. 2 is a graph showing Individual and Mean (±SD) Plasma TigecyclineConcentration in Sprague-Dawley Rats Following a Single Dose IV BolusInjection of 12 mg/kg.

FIG. 3 is a graph showing Dose Adjusted Mean Plasma TigecyclineConcentration in Sprague-Dawley Rats Following a Single Dose IV BolusInjection of 0.64 mg/kg or 12 mg/kg.

FIG. 4 is a graph showing Individual and Mean (±SD) Plasma TigecyclineConcentration in Sprague-Dawley Rats Following a Single Intraduodenal(ID) Injection formulated in PBS at 4.8 mg/kg.

FIG. 5 is a graph showing Individual and Mean (±SD) Plasma TigecyclineConcentration in Sprague-Dawley Rats Following a Single ID Injectionformulated in PBS at 9.0 mg/kg.

FIG. 6 is a graph showing Dose Adjusted Mean Plasma TigecyclineConcentration in Sprague-Dawley Rats Following a Single ID Injectionformulated in PBS.

FIG. 7 is a graph showing Individual and Mean (±SD) Plasma TigecyclineConcentration in Sprague-Dawley Rats Following a Single ID Injectionformulated in 100 mM citric acid (CA) (pH 3.5), 26 mMlauroyl-L-carnitine (LLC) at 4.8 mg/kg.

FIG. 8 is a graph showing Individual and Mean (±SD) Plasma TigecyclineConcentration in Sprague-Dawley Rats Following a Single ID Injectionformulated in 100 mM CA (pH 3.5), 26 mM LLC at 9.0 mg/kg.

FIG. 9 is a graph showing Dose Adjusted Mean Plasma TigecyclineConcentration in Sprague-Dawley Rats Following a Single ID InjectionFormulated in 100 mM CA (pH 3.5), 26 mM LLC.

FIG. 10 is a graph showing Individual and Mean (±SD) Plasma TigecyclineConcentration in Sprague-Dawley Rats Following a Single ID Injectionformulated in 400 mM CA (pH 3.5), 26 mM LLC at 4.8 mg/kg (using parentTigecycline peak only).

FIG. 11 is a graph showing Individual and Mean (±SD) Plasma TigecyclineConcentration in Sprague-Dawley Rats Following a Single ID Injectionformulated in 400 mM CA (pH 3.5), 26 mM LLC at 9.0 mg/kg (using parentTigecycline peak only).

FIG. 12 is a graph showing Dose Adjusted Mean Plasma TigecyclineConcentration in Sprague-Dawley Rats Following a Single ID InjectionFormulated in 400 mM CA (pH 3.5), 26 mM LLC (using parent Tigecyclinepeak only).

FIG. 13 is a graph showing Individual and Mean (±SD) Plasma TigecyclineConcentration in Sprague-Dawley Rats Following a Single ID Injectionformulated in 400 mM CA (pH 3.5), 26 mM LLC at 4.8 mg/kg (includingTigecycline-related peak).

FIG. 14 is a graph showing Individual and Mean (±SD) Plasma TigecyclineConcentration in Sprague-Dawley Rats Following a Single ID Injectionformulated in 400 mM CA (pH 3.5), 26 mM LLC at 9.0 mg/kg (includingTigecycline-related peak).

FIG. 15 is a graph showing Dose Adjusted Mean Plasma TigecyclineConcentration in Sprague-Dawley Rats Following a Single ID InjectionFormulated in 400 mM CA (pH 3.5), 26 mM LLC (includingTigecycline-related Peak).

FIG. 16 is a graph showing Mean Dose Adjusted Plasma TigecyclineConcentrations in Sprague-Dawley Rats Following Single ID Injections ofVarious Formulations from Primary Feasibility Studies RA851 and RA853.

FIG. 17 is a graph showing Study RA861—Mean (±SD) Plasma TigecyclineConcentrations in Sprague-Dawley Rats Following a Single ID Injection.

FIG. 18 is a graph showing Study RA869—Dose Adjusted Mean (±SD) PlasmaTigecycline Concentrations in Sprague-Dawley Rats.

FIG. 19 is a graph showing Study RA867—Mean (±SD) Plasma TigecyclineConcentrations in Sprague-Dawley Rats Following Single ID Administrationof Tigecycline Formulated in 400 mM CA, 26 mM LLC at pH 3.5 vs. pH 6.0.

FIG. 20 is a graph showing Mean Plasma Tigecycline Clearance Defined asPercent of Initial in Sprague-Dawley Rats Following Single IDAdministration (Studies RA851 and RA853).

FIG. 21 is a graph showing Individual PK Profiles for IV Formulation.

FIG. 22 is a graph showing Individual PK Profiles for Formulation A.

FIG. 23 is a graph showing Individual PK Profiles for Formulation PBS.

FIG. 24 is a graph showing Individual PK Profiles for Formulation B.

FIG. 25 is a graph showing Individual PK Profiles for Formulation C.

FIG. 26 is a graph showing Individual PK Profiles for Formulation D.

FIG. 27 is a graph showing Mean PK Profiles for Formulations ofZanamivir at the Indicated Dose.

FIG. 28 is a graph showing Mean Concentration (±SEM) Profiles vs TimeFollowing Bolus IV Injection of Kanamycin.

FIG. 29 is a graph showing Mean Concentration (±SEM) Profiles vs TimeFollowing Bolus IV Injection of Tobramycin.

FIG. 30 is a graph showing Individual Plasma Kanamycin AbsorptionProfiles for Dogs Following a Single Oral Dose in PROSOLV™ of UncoatedCapsules (Formulation JSV-003-038).

FIG. 31 is a graph showing Individual Plasma Kanamycin AbsorptionProfiles for Dogs Following a Single Oral Dose in Uncoated CapsulesFormulated with CA and LLC (Formulation JSV-003-039).

FIG. 32 is a graph showing Individual Plasma Profiles for Dogs Followinga Single Oral Dose of Capsules Containing 500 mg CA (FormulationJSV-003-005).

FIG. 33 is a graph showing Individual Plasma Profiles for Dogs Followinga Single Oral Dose of Capsules Containing 250 mg CA (FormulationJSV-003-052).

FIG. 34 is a graph showing Individual Plasma Profiles for Dogs Followinga Single Oral Dose of Capsules Containing 100 mg CA (FormulationJSV-003-053).

FIG. 35 is a graph showing Individual Plasma Profiles for Dogs Followinga Single Oral Dose of Capsules Containing 50 mg CA (FormulationJSV-003-054).

FIG. 36 is a graph showing Individual Plasma Profiles for Dogs Followinga Single Oral Dose of Capsules Containing 500 mg CA in DRCAPS™(Formulation JSV-003-010).

FIG. 37 is a graph showing Individual Plasma Profiles for Dogs Followinga Single Oral Dose of Capsules Containing 250 mg CA in DRCAPS™(Formulation JSV-003-041).

FIG. 38 is a graph showing Individual Plasma Profiles for Beagle DogsFollowing a Single Oral Dose in PROSOLV™ (Formulation JSV-003-050).

FIG. 39 is a graph showing Individual Plasma Profiles for Beagle DogsFollowing a Single Oral Dose with CA and LLC (Formulation JSV-003-051).

FIG. 40 is a graph showing Mean Absorption Profiles for DogsAdministered Kanamycin Formulated with 500 mg CA and 100 mg LLC inDRCAPS™ and Enteric-Coated VCAP PLUS™ Capsules.

FIG. 41 is a graph showing Mean Absorption Profiles for DogsAdministered Kanamycin in Unformulated and Formulated with CA and LLC inUncoated VCAP PLUS™ Capsules.

FIG. 42 is a graph showing Mean Absorption Profiles for DogsAdministered Kanamycin Formulated with 100 mg LLC and with VariousConcentrations of CA.

FIG. 43 is a graph showing Mean Absorption Profiles for DogsAdministered Unformulated and Formulated with CA and LLC CapsulesContaining Tobramycin.

FIG. 44 is a graph showing Individual and Mean (±SD) Plasma TigecyclineConcentration in Beagle Dogs Following a Single 1 mg (0.08 mg/kg) IVBolus Injection (SC427).

FIG. 45 is a graph showing Individual and Mean (±SD) Plasma TigecyclineConcentration in Beagle Dogs Following a Single 5 mg (0.42 mg/kg) IVBolus Injection (SC431).

FIG. 46 is a graph showing Dose Adjusted Mean Plasma TigecyclineConcentrations in Beagle Dogs Following Single IV Bolus Injections(SC427 and SC431).

FIG. 47 is a graph showing Individual Plasma Tigecycline Concentrationin Beagle Dogs Following a Single 15 mg (1.25 mg/kg) PO Enteric-CoatedCapsule Either Unformulated, or Formulated with 500 mg CA and 100 mg LLC(SC424).

FIG. 48 is a graph showing Mean (±SD) Plasma Tigecycline Concentrationin Beagle Dogs Following a Single 15 mg (1.25 mg/kg) PO Enteric-CoatedCapsule Either Unformulated, or Formulated with 500 mg CA and 100 mg LLC(SC424).

FIG. 49 is a graph showing Individual Plasma Tigecycline Concentrationin Beagle Dogs Following a Single 30 mg (2.5 mg/kg) PO Enteric-CoatedCapsule Formulated with 500 mg CA and 100 mg LLC (SC430).

FIG. 50 is a graph showing Mean (±SD) Plasma Tigecycline Concentrationin Beagle Dogs Following a Single 30 mg (2.5 mg/kg) PO Enteric-CoatedCapsule Formulated with 500 mg CA and 100 mg LLC (SC430).

FIG. 51 is a graph showing Individual Plasma Tigecycline Concentrationin Beagle Dogs Following a Single 45 mg (3.75 mg/kg) PO Enteric-CoatedCapsule Formulated with 500 mg CA and 100 mg LLC (SC430).

FIG. 52 is a graph showing Mean (±SD) Plasma Tigecycline Concentrationin Beagle Dogs Following a Single 45 mg (3.75 mg/kg) PO Enteric-CoatedCapsule Formulated with 500 mg CA and 100 mg LLC (SC430).

FIG. 53 is a graph showing Mean Plasma Tigecycline Concentrations inBeagle Dogs Following a Single PO Enteric-Coated Capsule Formulated with500 mg CA and 100 mg LLC (SC424 and SC430). (Note the time scale hasbeen adjusted for comparison as SC424 sampled to 4 hours, while SC430sampled to 24 hours).

FIG. 54 is a graph showing Mean Dose Adjusted Plasma TigecyclineConcentrations in Beagle Dogs Following a Single PO Enteric-CoatedCapsule Formulated with 500 mg CA and 100 mg LLC (SC424 and SC430). Notethe time scale has been adjusted for comparison as SC424 sampled to 4hours, while SC430 sampled to 24 hours.

FIG. 55 is a graph showing Dose Linearity With Respect to C_(max) inBeagle Dogs Following a Single PO Enteric-Coated Capsules Formulatedwith 500 mg CA and 100 mg LLC. Note the time scale has been adjusted forcomparison as SC424 sampled to 4 hours, while SC430 sampled to 24 hours.

FIG. 56 is a graph showing Dose Linearity With Respect to AUC_((0-4HR))in Beagle Dogs Following a Single PO Enteric-Coated Capsules Formulatedwith 500 mg CA and 100 mg LLC. Note the time scale has been adjusted forcomparison as SC424 sampled to 4 hours, while SC430 sampled to 24 hours.

FIG. 57 is a graph showing Solubility of Fenofibrate with increasingconcentrations of LLC.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a pharmaceutical composition suitable fororal delivery including at least one compound classified as BCS ClassII, BCS Class III or BCS Class IV; at least one absorption enhancer; atleast one pH lowering compound; and at least one chelating agent.

The at least one compound classified as BCS Class II, BCS Class III orBCS Class IV can be a small molecule organic compound. The at least onecompound classified as BCS Class II, BCS Class III or BCS Class IV canbe an antibiotic or antiviral compound. The at least one compoundclassified as BCS Class II, BCS Class III or BCS Class IV can be atigecycline, zanamivir, kanamycin, tobramycin or fenofibrate.

The pharmaceutical composition can be a solid dosage pharmaceuticalcomposition (e.g., tablet, capsule). The pharmaceutical composition canbe a multilayer solid dosage pharmaceutical composition.

The pH lowering compound can have a pKa no higher than 4.2, the pHlowering compound can have a pKa no higher than 3.0. Preferably, the pHlowering agent is sodium citrate.

The chelating agent can be a carboxylic acid chelating agent or an aminoacid chelating agent. The carboxylic acid chelating agent can beacetylsalicylic acid, acetic acid, ascorbic acid, citric acid, fumaricacid, glucuronic acid, glutaric acid, glyceric acid, glycocolic acid,glyoxic acid, isocitric acid, isovaleric acid, lactic acid, maleic acid,oxaloacetic acid, oxalosuccinic acid, propionic acid, pyruvic acid,succinic acid, tartaric acid, or valeric acid. Preferably, thecarboxylic acid chelating agent is citric acid.

The at least one absorption enhancer can include an acylcarnitine.Preferably, the acylcarnitine is lauroyl carnitine. The at least oneabsorption enhancer can include a surface acting agent. The surfaceacting agent can be an acid soluble bile acid.

The pharmaceutical composition can further include a cationic surfaceacting agent.

The pharmaceutical composition can further include an acid resistantprotective vehicle. Preferably, the acid resistant protective vehicle isa viscous protective syrup.

A multilayered solid dosage pharmaceutical composition including achelating agent and an acid resistant protective vehicle can alsoinclude a water soluble barrier is layered between the chelating agentand the acid resistant protective vehicle.

The present invention provides a pharmaceutical composition suitable fororal delivery including: at least one antibiotic or antiviral compoundclassified as BCS Class II, BCS Class III or BCS Class IV;lauroyl-carnitine; citric acid; and sodium citrate, wherein thecomposition is buffered at pH 3.5.

The present invention provides pharmaceutical compositions comprising atherapeutically effective amount of at least one compound selected fromone of a Class II drug, a Class III drug or a Class IV drug; at leastone chelating agent; and at least one absorption enhancer. Thecomposition can further comprise at least one pH lowering agent. Thecomposition can further comprise an acid resistant protective vehicleeffective to transport the pharmaceutical composition through thestomach of a patient. The composition can further comprise a watersoluble barrier layer that separates the chelator from the acidresistant protective vehicle. In an aspect, the chelating agent is acarboxylic acid. In an aspect, the carboxylic acid is citric acid. In anaspect, the citric acid is present in the pharmaceutical composition ina quantity which, if the composition were added to ten milliliters of0.01M aqueous sodium bicarbonate solution, would be sufficient to lowerthe pH of the solution to no higher than 5.5. In an aspect, the citricacid is present in the pharmaceutical composition in a quantity which,if the composition were added to ten milliliters of 0.05M aqueous sodiumbicarbonate solution, would be sufficient to lower the pH of thesolution to no higher than 5.5. In an aspect, the citric acid is presentin the pharmaceutical composition in a quantity which, if thecomposition were added to ten milliliters of 0.1M aqueous sodiumbicarbonate solution, would be sufficient to lower the pH of thesolution to no higher than 5.5. In an aspect, the absorption enhancer islauroyl carnitine.

Pharmaceutical Compositions

The present invention provides a pharmaceutical composition suitable fororal delivery including at least one compound classified as BCS ClassII, BCS Class III or BCS Class IV.

The Biopharmaceutical Classification System (BCS), originally developedby G. Amidon, separates pharmaceuticals for oral administration intofour classes depending on their aqueous solubility and theirpermeability through the intestinal cell layer. According to the BCS,drug substances are classified as follows: Class I—High Permeability,High Solubility; Class II—High Permeability, Low Solubility; ClassIII—Low Permeability, High Solubility; and Class IV—Low Permeability,Low Solubility.

As used herein, a compound is considered highly soluble when the highestdose strength is soluble in <250 ml water over a pH range of 1 to 7.5.As used herein, a compound is considered highly permeable when theextent of absorption in humans is determined to be >90% of anadministered dose, based on mass-balance or in comparison to anintravenous reference dose. As used herein, a compound is considered tobe rapidly dissolving when >85% of the labeled amount of drug substancedissolves within 30 minutes using USP apparatus I or II in a volume of<900 ml buffer solutions.

As used herein, a compound or drug (these terms are interchangeably)does not include a peptide bond in its molecular structure. It should beunderstood that a compound of the invention can comprise a smallmolecule. The term small molecule as used herein refers to a lowmolecular weight organic, inorganic, or organometallic compound. A smallmolecule may comprise a molecular weight of less than 2000 Daltons. Asmall molecule may comprise a molecular weight of less than 500 Daltons.A small molecule may comprise a molecular weight of about 50 to 500Daltons.

A compound of the present invention can be a compound that targetsbacterial functions or growth processes, for example an antibiotic. Thecompound can be an antibiotic that contains a central four-ringcarbocyclic skeleton. The antibiotic can be a tetracycline orglycylcycline antibiotic. In preferred aspects, the antibiotic istigecycline. The compound can be capable of binding to a ribosomalsubunit of a bacterium. A compound of the present invention can be acompound that targets a virus or viral particle, for example anantiviral agent or compound.

The compound can be classified as a BCS class II drug, a class III drug,or a BCS class IV drug. Non-limiting examples of BCS class II drugs are:glibenclamide, bicalutamide, ezetimibe, fenofibrate, glipizide,atovaquone, carbamazepine, danazol, griseofulvin, ketoconazole,toglitazone, ibuprofen, nifedipine, nitrofurantoin, phenytoin,sulfamethoxazole, trimethoprim, valproic acid, praziquantel, retinolpalmitate, and sulfasalazine. Non-limiting examples of BCS class IIIdrugs are: cimetidine, acyclovir, atenolol, ranitidine, abacavir,captopril, chloramphenicol, codeine, colchicine, dapsone, ergotamine,kanamycin, tobramycin, tigecycline, zanamivir, hydralazine,hydrochlorothiazide, levothyroxine, methyldopa, paracetamol,propylthiouracil, pyrodostigmine, sodium cloxacillin, thiamine,benzidazole, didanosine, ethambutol, ethosuximide, folic acid,nicotinamide, nifurtimox, and salbutamol sulfate. Non-limiting examplesof BCS class IV drugs are: hydrochlorothiazide, furosemide, cyclosporinA, itraconazole, indinavir, nelfinavir, ritonavir, saquinavir,nitrofurantoin, albendazole, acetazolamide, azithromycin.

In some preferred aspects of the present invention, the compound isTigecycline. Tigecycline is the first approved member in a new class ofglycylcycline-based tetracycline antibiotics. Tigecycline exhibitsactivity against a variety of gram-positive and gram-negative bacterialpathogens, many of which are resistant to existing antibiotics—includingactivity against Methicillin-Resistant Staphylococcus aureus (MRSA),Stenotrophomonas maltophilia, Haemophilus influenzae, and Neisseriagonorrhoeae (with MIC values reported at 2 mcg/mL) and multi-drugresistant strains of Acinetobacter baumannii, as non-limiting examples.Tigecycline is licensed for the treatment of skin and soft tissueinfections as well as intra-abdominal infections and has been previouslyutilized as a lyophilized powder for reconstitution for IV infusion inthe hospital setting primarily due to its inherently low innatepermeability. Tigecycline's aqueous solubility is approximately 300mg/mL, its permeability liability makes oral administration a challenge.Known formulations exhibit maximal oral bioavailablity % (% F) less than5%. Commensurate with its high aqueous solubility and poor membranepermeation, tigecycline is not extensively metabolized. The drug isprimarily cleared through the biliary route, largely as unchanged drug.In one aspect, the pharmaceutical composition of the present inventionis an improved oral dosage formula of tigecycline. In one aspect, thepharmaceutical composition of the present invention is an oral dosageformula of tigecycline for oral conversion of treatment after apatient's clinical signs have stabilized, indicating control ofinfection. In one aspect, the oral dosage formulation of tigecycline ofthe present invention is used to control recurrent infections inpatients with no, or minimal hepatic impairment.

In some preferred aspects of the present invention, the compound iszanamivir. In one aspect, a pharmaceutical composition of the presentdisclosure is zanamivir formulated with a pH-lowering agent (e.g.buffered citric acid) and/or a permeation enhancer (e.g.lauroyl-L-carnitine) or as an emulsion (e.g. an emulsion in Capmul).

A pharmaceutical composition suitable for oral delivery including atleast one compound classified as BCS Class II, BCS Class III or BCSClass IV of the present invention can also comprise at least onechelating agent. The chelating agent can be a carboxylic acid chelatingagent or an amino acid chelating agent.

Suitable carboxylic acids that can be used as a chelating agent of thepresent disclosure, include, but are not limited to, acetylsalicylic,acetic, ascorbic, citric, fumaric, glucuronic, glutaric, glyceric,glycocolic, glyoxylic, isocitric, isovaleric, lactic, maleic,oxaloacetic, oxalosuccinic, propionic, pyruvic, succinic, tartaric,valeric, and the like.

Suitable organic amino acids that can be used as a chelating agent ofthe present disclosure, include, but are not limited to, glutamic acid,aspartic acid, histidine, and the like.

The chelating agent can be a high affinity chelating agent. A highaffinity chelating agent can chelate cationic metals, thereby inhibitingsalt-induced precipitation. In a preferred aspect, the chelating agentis citric acid. Citric acid exhibits three (3) ionizable groups,distinguished by 3 different pKa, circa pH=3.09, 4.75, and 6.39. Thisproperty allows citric acid to act as a polydentate binder, orsequestering agent of cationic species in solution. The net result isthat citric acid is an excellent chelating agent, specifically for smallmetal ions such as calcium. In the active pH range of the currentformulation (<pH 5.5) however because of the respective pKa of theionizable groups, one would expect that the larger percentage of 1, or 2(depending on pH) of these ionizable groups would be protonated, andtherefore, not available to bind cationic salts, such as calcium. Assuch, at the pH in question (<pH 5.5), citric acid would not be expectedto be as efficient a chelating agent in this pH range as in more basicconditions.

Suitable chelating agents can also include EDTA, EGTA, Phosphonates, andbisphosphonates.

A pharmaceutical composition suitable for oral delivery including atleast one compound classified as BCS Class II, BCS Class III or BCSClass IV of the present invention can also comprise coated acidparticles.

In one aspect, the carboxylic acid can be provided, at least in part, byacid particles coated with a protective coating to reduce undesirableacid interaction with other components of the formulation, such as thecompound and, where used, the outer enteric coating. When coated acidparticles are used, the particles are coated with a pharmaceuticallyacceptable protective coating that is non-acidic and preferably has asolubility in water of at least one gram, and preferably at least 10grams, per 100 milliliters of water at room temperature. As the coatingis for the purpose of reducing acid interaction with other components ofthe pharmaceutical composition, it is important that the coating notitself be acidic such that its own acidity could undesirably cause someof the acid interactions that it is the coating's purpose to prevent.Good water solubility is also important for quick dissolution, which inturn desirably aids a more simultaneous release of the pharmaceuticalacid, the drug and the absorption enhancer.

Appropriate coating materials include but are not limited to sugars(e.g. glucose), oligosaccharides (maltodextrin), and acid salts (e.g.sodium citrate). When acid salts are used, it is preferred, but notrequired, that they be salts of the acid being coated (e.g., sodiumcitrate-coated citric acid particles). Preferred coated acid particlesinclude but are not limited to maltodextrin-coated citric acid particlesavailable from Jungbunzlauer under the trademark CITROCOAT. When used asthe acid, citric acid or other organic acids can be coated by spraying acoating solution which contains, for example, glucose, maltodextrin orsodium citrate onto granules of an organic acid in a fluid-bed dryer.Coatings discussed herein may be used on particles of other acidsdiscussed herein.

The average size of the acid-coated particles can be from about 30 meshto about 140 mesh.

A pharmaceutical composition suitable for oral delivery including atleast one compound classified as BCS Class II, BCS Class III or BCSClass IV of the present invention can also comprise at least onepH-lowering compound.

The quantity of pH-lowering compound can be determined based on the typeof pH-lowering compound used and the equivalents of protons provided bya given pH-lowering compound.

The pH-lowering compound can be any pharmaceutically acceptable compoundthat is not toxic in the gastrointestinal tract and is capable of eitherdelivering hydrogen ions (a Bronsted-Lowry acid) or of inducing higherhydrogen ion content from the local environment (acting as an Arrheniusor Lewis acid). It can also be any combination of such compounds and/ora combination of such compounds and their respective conjugate bases tomaintain the target pH. In one aspect, at least one pH-lowering compoundused in the composition has a pKa no higher than 4.2, and preferably nohigher than 3.0. In one aspect, at least one pH-lowering compound has asolubility in water of at least 30 grams per 100 milliliters of water atroom temperature.

Non-limiting examples of compounds that are Arrhenius or Lewis acidsinclude halide salts of metals, such as aluminum chloride and zincchloride. Pharmaceutically acceptable traditional acids include, but arenot limited to acid salts of amino acids (e.g., amino acidhydrochlorides) or derivatives thereof. Examples of these are acid saltsof acetylglutamic acid, alanine, arginine, asparagine, aspartic acid,betaine, carnitine, carnosine, citrulline, creatine, glutamic acid,glycine, histidine, hydroxylysine, hydroxyproline, hypotaurine,isoleucine, leucine, lysine, methylhistidine, norleucine, ornithine,phenylalanine, proline, sarcosine, serine, taurine, threonine,tryptophan, tyrosine and valine.

Other useful pH-lowering compounds that might not usually be called“acids” in the art, but which may nonetheless be useful in accordancewith the invention are organophosphates with at least one freephosphohydroxyl group, such as phosphate esters (e.g., fructose 1, 6diphosphate, glucose 1, 6 diphosphate, phosphoglyceric acid, anddiphosphoglyceric acid). CARBOPOL™. (Trademark BF Goodrich) and polymerssuch as polycarbophil may also be used to lower pH.

A pharmaceutical composition suitable for oral delivery including atleast one compound classified as BCS Class II, BCS Class III or BCSClass IV of the present invention can also comprise at least oneabsorption enhancer.

The absorption enhancers can be present in a quantity that constitutesfrom 0.1 to 20.0 percent by weight, relative to the overall weight ofthe pharmaceutical composition (exclusive of the enteric coating).Suitable absorption enhancers can be surface active agents which actboth as solubility enhancers and uptake enhancers. Generically speaking,“solubility enhancers” improve the ability of the components of thepresent disclosure to be solubilized in either the aqueous environmentinto which they are originally released or into the lipophilicenvironment of the mucous layer lining the intestinal walls, or both.“Transport (uptake) enhancers” (which are frequently the same surfaceactive agents used as solubility enhancers) are those which facilitatethe ease by which drugs cross the intestinal wall.

One or more absorption enhancers may perform one function only (e.g.,solubility), or one or more absorption enhancers may perform the otherfunction only (e.g., uptake). It is also possible to have a mixture ofseveral compounds some of which provide improved solubility, some ofwhich provide improved uptake and/or some of which perform both. Withoutintending to be bound by theory, it is believed that uptake enhancersmay act by (1) increasing disorder of the hydrophobic region of themembrane exterior of intestinal cells, allowing for increasedtranscellular transport; or (2) leaching membrane proteins resulting inincreased transcellular transport; or (3) widening pore radius betweencells for increased paracellular transport.

Surface active agents can be useful both as solubility enhancers and asuptake enhancers. For example, detergents are useful in (1) solubilizingall of the active components quickly into the aqueous environment wherethey are originally released, (2) enhancing lipophilicity of thecomponents of the present disclosure, especially the drug, aiding itspassage into and through the intestinal mucus, (3) enhancing the abilityof the drug to cross the epithelial barrier of the brush bordermembrane; and (4) increasing transcellular or paracellular transport asdescribed herein.

When surface active agents are used as the absorption enhancers, theycan be free flowing powders for facilitating the mixing and loading ofcapsules during the manufacturing process. When trying to increase thebioavailability of a compound, the surface active agent used as anabsorption enhancer can be selected from the group consisting of (i)anionic surface active agents that are cholesterol derivatives (e.g.,bile acids), (ii) cationic surface agents (e.g., acyl carnitines,phospholipids and the like), (iii) non-ionic surface active agents, and(iv) mixtures of anionic surface active agents (especially those havinglinear hydrocarbon regions) together with negative charge neutralizers.Negative charge neutralizers include but are not limited to acylcarnitines, cetyl pyridinium chloride, and the like. The absorptionenhancer can be soluble at acid pH, particularly in the 3.0 to 5.0range.

In one aspect, a combination of a cationic surface active agent togetherwith an anionic surface active agent can be present in a pharmaceuticalcomposition of the present invention. In one aspect, both the cationicsurface active agent and the anionic surface active agent can becholesterol derivatives and both can be soluble at acid pH.

In one aspect, a combination of an acid soluble bile acid together witha cationic surface active agent can be present in a pharmaceuticalcomposition of the present invention. In one aspect, the combination canbe acyl carnitine and sucrose ester. When a particular absorptionenhancer is used alone, it is preferred that it be a cationic surfaceactive agent. Acyl carnitines (e.g., lauroyl carnitine), phospholipidsand bile acids are particularly good absorption enhancers, especiallyacyl carnitine. Anionic surfactants that are cholesterol derivatives arealso used in some aspects. It is the intent of these preferences toavoid interactions with the drug that interfere with absorption of thedrug into the blood.

To reduce the likelihood of side effects, preferred detergents, whenused as the absorption enhancers of the present disclosure, can beeither biodegradable or reabsorbable (e.g. biologically recyclablecompounds such as bile acids, phospholipids, and/or acyl carnitines),preferably biodegradable. Acylcarnitines are believed particularlyuseful in enhancing paracellular transport.

Non-limiting examples of absorption enhancers include: (a) salicylatessuch as sodium salicylate, 3-methoxysalicylate, 5-methoxysalicylate andhomovanilate; (b) bile acids such as taurocholic, tauorodeoxycholic,deoxycholic, cholic, glycholic, lithocholate, chenodeoxycholic,ursodeoxycholic, ursocholic, dehydrocholic, fusidic, etc.; (c) non-ionicsurfactants such as polyoxyethylene ethers (e.g. Brij 36T, Brij 52, Brij56, Brij 76, Brij 96, Texaphor A6, Texaphor A14, Texaphor A60 etc.),p-t-octyl phenol polyoxyethylenes (Triton X-45, Triton X-100, TritonX-114, Triton X-305 etc.) nonylphenoxypoloxyethylenes (e.g. Igepal COseries), polyoxyethylene sorbitan esters (e.g. Tween-20, Tween-80 etc.),d-alpha tocopheryl polyethylene glycol 1000 succinate (vitamin E TPGS);(d) anionic surfactants such as dioctyl sodium sulfosuccinate; (e)lyso-phospholipids such as lysolecithin andlysophosphatidylethanolamine; (f) acylcarnitines, acylcholines and acylamino acids such as lauroylcarnitine, myristoylcarnitine,palmitoylcarnitine, lauroylcholine, myristoylcholine, pahnitoylcholine,hexadecyllysine, N-acylphenylalanine, N-acylglycine etc.; (g) watersoluble phospholipids such as diheptanoylphosphatidylcholine,dioctylphosphatidylcholine etc.; (h) medium-chain glycerides which aremixtures of mono-, di- and triglycerides containing medium-chain-lengthfatty acids (caprylic, capric, lauric acids and the like); (i)ethylene-diaminetetraacetic acid; (j) cationic surfactants such ascetylpyridinium chloride, benzalkonium chloride, benzethonium chlorideand the like; (k) fatty acid derivatives of polyethylene glycol such asLabrasol, Labrafac, etc.; and (l) alkylsaccharides such as laurylmaltoside, lauroyl sucrose, myristoyl sucrose, palmitoyl sucrose, etc.In one aspect, the absorption enhancer is lauroyl carnitine.

A pharmaceutical composition suitable for oral delivery including atleast one compound classified as BCS Class II, BCS Class III or BCSClass IV of the present invention can also comprise an acid-resistantprotective vehicle.

Many acid-resistant protective vehicles (enteric coatings) are known inthe art, and are useful in accordance with the present disclosure.Examples include cellulose acetate phthalate, hydroxypropylmethylethylcellulose succinate, hydroxypropyl methylcellulose phthalate,carboxyl methylethylcellulose and methacrylic acid-methyl methacrylatecopolymer. In one aspect, the compound, absorption enhancers such assolubility and/or uptake enhancer(s), and carboxylic acids, are includedin a sufficiently viscous protective syrup to permit protected passageof the components of the present disclosure through the stomach.

Suitable enteric coatings may be applied, for example, to capsules afterthe remaining components of the present disclosure have been loadedwithin the capsule. In other aspects, enteric coating is coated on theoutside of a tablet or coated on the outer surface of particles ofactive components which are then pressed into tablet form, or loadedinto a capsule, which is itself preferably coated with an entericcoating.

All components of the present disclosure should be released from thecarrier or vehicle, and solubilized in the intestinal environment assimultaneously as possible. It is preferred that the vehicle or carrierrelease the active components in the small intestine where uptakeenhancers that increase transcellular or paracellular transport are lesslikely to cause undesirable side effects than if the same uptakeenhancers were later released in the colon. It is emphasized, however,that the present disclosure is believed effective in the colon as wellas in the small intestine. Numerous vehicles or carriers, in addition tothe ones discussed above, are known in the art. It is desirable(especially in optimizing how simultaneously the components of thepresent disclosure are released) to keep the amount of enteric coatinglow. Preferably, the enteric coating adds no more than 30% to the weightof the remainder of pharmaceutical composition (the “remainder” beingthe pharmaceutical composition exclusive of enteric coating itself).More preferably, it adds less than 20%, especially from 12% to 20% tothe weight of the uncoated composition. The enteric coating preferablyshould be sufficient to prevent breakdown of the pharmaceuticalcomposition of the present disclosure in 0.1N HCl for at least twohours, then capable of permitting complete release of all contents ofthe pharmaceutical composition within thirty minutes after pH isincreased to 6.3 in a dissolution bath in which said composition isrotating at 100 revolutions per minute. In aspects in which thewater-soluble barrier layer of the present disclosure is used, lessenteric coating may be required, sometimes less that the amount ofwater-soluble barrier layer.

A pharmaceutical composition suitable for oral delivery including atleast one compound classified as BCS Class II, BCS Class III or BCSClass IV of the present invention can be a solid dosage form. Thepharmaceutical composition can also be a multi-layered solid dosageform.

In an aspect where the pharmaceutical composition can also be amulti-layered solid dosage form, and the pharmaceutical compositionincludes citric acid and an acid resistant protective vehicle, awater-soluble barrier can be present to separate the chelating agentfrom the acid resistant protective vehicle. In some of the exampleswhich follow, a conventional pharmaceutical capsule is used for thepurpose of providing this barrier. Many water soluble barriers are knownin the art and include, but are not limited to, hydroxypropylmethylcellulose and conventional pharmaceutical gelatins.

In an aspect, a peptide (such as albumin, casein, soy protein, otheranimal or vegetable proteins and the like) can be included to reducenon-specific adsorption (e.g., binding of peptide to the intestinalmucus barrier). When added, the peptide is preferably from 1.0 to 10.0percent by weight relative to the weight of the overall pharmaceuticalcomposition (excluding protective vehicle). Preferably, the peptide isnot physiologically active and is most preferably a food peptide such assoy bean peptide or the like.

All pharmaceutical compositions of the present disclosure can alsocomprise common pharmaceutical diluents, glidants, fillers, lubricants,antioxidants, gelatin capsules, preservatives, colorants and the like intheir usual known sizes and amounts.

A suitable filler includes a cellulose filler like PROSOLV™ availablefrom JRS Pharma be utilized. Other fillers are known in the art can alsobe utilized.

Any disintegrant that performs the function of enhancing dissolutionspeed may be used. Non-limiting examples of suitable disintegrantsinclude POLYPLASDONE, EXPLOTAB, and AC-DI-SOL, available fromInternational Specialty Products, JRS Pharma and FMC Biopolymer,respectively. Preferably, the disintegrant is present in an amountbetween 1 and 15 percent by weight relative to the total tablet weight(when tablets are used), exclusive of any water-soluble barrier layerand any acid-resistant protective vehicle.

Any glidant that performs the function of enhancing powder flow may beused. Non-limiting examples of suitable glidants include talc, calciumsilicate, magnesium silicate, silicon dioxide. Preferably, the glidantis present in an amount between 0.1 and 2.0 percent by weight relativeto the weight of the pharmaceutical composition, exclusive of anywater-soluble barrier layer and any acid-resistant protective vehicle.

Any lubricant that performs the function of preventing powder fromsticking to the tooling may be used. Non-limiting examples of suitablelubricants include stearic acid, magnesium stearate, and hydrogenatedvegetable oil type 1. Preferably, the lubricant is present in an amountbetween 0.5 and 5.0 percent by weight relative to the weight of thepharmaceutical composition, exclusive of any water-soluble barrier layerand any acid-resistant protective vehicle.

Non-limiting examples of suitable antioxidants include sodium pyruvate,derivatives of sodium pyruvate, ascorbic acid, ascorbyl palmitate,butylated hydroxyanisole, butylated hydroxytoluene, sodium bisulfate,and sodium metabisulfite. Preferably, the antioxidant is present in anamount between 0.5 and 5 mg per tablet.

The pharmaceutical composition can also comprise a peptide (such asalbumin, casein, soy protein, other animal or vegetable proteins and thelike) to reduce non-specific adsorption (e.g., binding of peptide to theintestinal mucus barrier) thereby lowering the necessary concentrationof the drug. When added, the peptide is preferably from 1.0 to 10.0percent by weight relative to the weight of the overall pharmaceuticalcomposition (excluding any water-soluble barrier layer and anyacid-resistant protective vehicle). Preferably, the peptide is notphysiologically active and is most preferably a food peptide such assoybean peptide or the like.

The pharmaceutical composition can be a solid dosage form. Once suitablesolid dosage form is a tablet. When the pharmaceutical composition is atable a pharmaceutical binder for dry compression can also be included.Non-limiting examples of suitable binders include KOLLIDON VA64,KOLLIDON VA64 fine, KOLLIDON 30, AVICEL PH-101, PHARMACOAT 606, andMALDEX. The first three are commercially available from BASF, and thelatter three are available from FMC Biopolymer, Shin-Etsu, and Amylum,respectively.

To improve simultaneous release, thorough intermixing of the componentsof the pharmaceutical composition (other than any optional entericcoating or barrier layer) results in substantially uniform dispersion ofsaid components within the binder. For this purpose, coated acidparticles (when used) are considered a single component. It isespecially preferred that acid (or when used, coated acid particles) anddrug be uniformly dispersed.

When prepared in tablet form, it is preferred that the maximum weightloss during friability testing be no greater than 1%. As used herein,friability testing refers to the technique described in “TabletFriability”, Chapter 1216, USP 28 page 2745.

In one aspect, the weight ratio of citric acid to absorption enhancercan be between 3:1 and 20:1, preferably 4:1-12:1, and most preferably5:1-10:1. The total weight of citric acid and the total weight ofabsorption enhancer in a given pharmaceutical composition are includedin the foregoing preferred ratios.

In one aspect, a compound, a pH lowering compound, and a absorptionenhancer (whether single compounds or a plurality of compounds in eachcategory) are uniformly dispersed in the pharmaceutical composition. Thepharmaceutical composition can comprise granules that include apharmaceutical binder having the drug, the citric acid and theabsorption enhancer uniformly dispersed within said binder. Preferredgranules may also consist of an acid core, surrounded by a uniform layerof organic acid, a layer of enhancer and a layer of drug that issurrounded by an outer layer of organic acid. Granules may be preparedfrom an aqueous mixture consisting of pharmaceutical binders such aspolyvinyl pyrrolidone or hydroxypropyl methylcellulose, together withthe citric acid, absorption enhancer and drug of the present disclosure,or by dry granulation processes. Other aspects include matrix or othertablet or capsule-based systems that may include multiple granulationphases, such as granulation of the drug, with or without solubilityenhancing excipients and other processing excipients, in concert with anexternal granulated citric acid phase.

Solubility enhancement for compounds with low or no water solubility canalso be achieved using additional processing techniques known in theart. Such techniques include, but are not limited, to spray drying,lyophilization, or hot melt extrusion to form an amorphic dispersionusing a suitable, pharmaceutically acceptable polymer, such as PVP,HPMC, HPMCP, HPMCAS, PEG, PVP/VA, MME, CAP, polaxamer, gelucire, Tween,Eudragit, CMEC, gelatin, etc. Many other such additional excipients willbe apparent to one of skill in the art. The permeation enhancer can beused as the surfactant to aid dispersion formation during processing,while maintaining its permeation enhancing affects in vivo. Such aspectscould then be filled as a dry blend into capsules with the remainingactive and processing excipients, or compressed into tablets, with orwithout granulation.

In an aspect, a pharmaceutical composition of the present inventioncomprises a size 00 gelatin or HPMC capsule filled with 50 mg ofcompound, 500 mg of granular citric acid (available for example fromArcher Daniels Midland Corp.), and 50 mg lauroyl carnitine (SIGMA).

All of the ingredients are preferably for eventual insertion into thegelatin or HPMC capsule and are preferably powders which may be added toa blender in any order. Thereafter, the blender is run for about fiveminutes until the powders are thoroughly intermixed. Then the mixedpowders are loaded into the large end of the capsules. The other end ofthe capsule is then added, and the capsule snapped shut. 500 or moresuch capsules may be added to a coating device (e.g., Vector LDCS 20/30Laboratory Development Coating System (available from Vector Corp.,Marion, Iowa)).

An enteric coating solution is made as follows. Weigh 500 grams ofEUDRAGIT L30 D-55 (poly(methacrylic acid-co-ethyl acrylate) 1:1; amethacrylic acid-ethyl acrylate copolymer (1:1), an enteric coatingavailable from Evonik). Add 411 grams distilled water, 15 grams triethylcitrate and 38 grams talc. This amount of coating will be sufficient tocoat about 500 size 00 capsules.

The capsules are the film coated using processes known in the art.

Because of the enhanced bioavailability provided by the presentdisclosure, the concentration of expensive drug in the pharmaceuticalpreparation of the present disclosure may be kept relatively low.

In an aspect, a pharmaceutical composition of the present disclosure isa tablet prepared as follows:

1. High shear or COMIL™ geometrical mixing of drug and microcrystallinecellulose (PROSOLV™—such as PROSOLV™ HD90).2. Add mixed components of step 1 to V blender along with citric acid DCF20, lauroyl-L-carnitine, Crospovidone, KOLLIDON VA64 and sodiumpyruvate. Mix in V blender.3. Add magnesium stearate to V blender after step 2 completed. Mix in Vblender briefly.4. Compress blend into tablets.5. Coat tablets with optional water-soluble barrier, such ashydroxypropylmethylcellulose (HPMC) or PVA subcoat, to 6% weight gain.6. Coat tablets with optional enteric coat (EUDRAGIT L30D-55) to 7%weight gain.

Methods of Treatment

The present invention also provides methods for enhancing thebioavailability of a therapeutically effective amount of at least onecompound classified as BCS Class II, BCS Class III or BCS Class IVincluding orally administering a pharmaceutical composition including atleast one compound classified as BCS Class II, BCS Class III or BCSClass IV; at least one absorption enhancer; at least one pH loweringcompound; and at least one chelating agent.

The present invention also provides methods of treating a bacterial orviral infection in a subject in need thereof including orallyadministering a pharmaceutical composition including at least onecompound classified as BCS Class II, BCS Class III or BCS Class IV; atleast one absorption enhancer; at least one pH lowering compound; and atleast one chelating agent. The bacterial infection can be agram-positive or gram-negative infection.

The present invention also provides methods of treating complicated skinand skin structure infections (cSSSI) in a subject in need thereofincluding orally administering a pharmaceutical composition including atleast one compound classified as BCS Class II, BCS Class III or BCSClass IV; at least one absorption enhancer; at least one pH loweringcompound; and at least one chelating agent.

The terms “subject” and “patient,” as used herein, describes anorganism, including mammals, to which treatment with the compositionsand methods of the present invention are provided. A “subject” includesa mammal. The mammal can be e.g., any mammal, e.g., a human, primate,bird, mouse, rat, fowl, dog, cat, cow, horse, goat, camel, sheep or apig. Preferably, the mammal is a human. The terms “subject” and“patient” are used interchangeable herein.

The term “therapeutically effective amount,” as used herein, refers toan amount of a pharmaceutical composition to treat, ameliorate, orprevent an identified disease or condition, or to exhibit a detectabletherapeutic or inhibitory effect. The effect can be detected by anyassay method known in the art. The precise effective amount for asubject will depend upon the subject's body weight, size, and health;the nature and extent of the condition; and the therapeutic orcombination of therapeutics selected for administration.

According to aspects illustrated herein, there is disclosed a method forenhancing the bioavailability of a Class II, III or IV drug deliveredorally that includes selectively releasing a therapeutically effectiveamount of the drug, together with at least one chelating agent and atleast one absorption enhancer, into a patient's intestine followingpassage of the drug, chelating agent and absorption enhancer through thepatient's mouth and stomach.

According to aspects illustrated herein, there is disclosed an oralpharmaceutical composition that includes a therapeutically effectiveamount of tigecycline; a sufficient amount of citric acid to yieldchelating properties; and lauroyl carnitine. In an aspect, thecomposition further comprises at least one pH lowering agent. In anaspect, the composition further comprises an acid resistant protectivevehicle effective to transport the pharmaceutical composition throughthe stomach of a patient. In an aspect, the composition furthercomprises a water soluble barrier layer that separates the citric acidfrom the acid resistant protective vehicle. In an aspect, the citricacid is present in the pharmaceutical composition in a quantity which,if the composition were added to ten milliliters of 0.01M aqueous sodiumbicarbonate solution, would be sufficient to lower the pH of thesolution to no higher than 5.5. In an aspect, the citric acid is presentin the pharmaceutical composition in a quantity which, if thecomposition were added to ten milliliters of 0.05M aqueous sodiumbicarbonate solution, would be sufficient to lower the pH of thesolution to no higher than 5.5. In an aspect, the citric acid is presentin the pharmaceutical composition in a quantity which, if thecomposition were added to ten milliliters of 0.1M aqueous sodiumbicarbonate solution, would be sufficient to lower the pH of thesolution to no higher than 5.5.

According to aspects illustrated herein, there is disclosed a method forenhancing the bioavailability of tigecycline delivered orally thatincludes selectively releasing a therapeutically effective amount oftigecycline, together with a sufficient amount of citric acid andlauroyl carnitine, into a patient's intestine following passage of thetigecycline, citric acid, and lauroyl carnitine through the patient'smouth and stomach.

The simultaneous use of absorption enhancers together with a chelatingagent, in accordance with the present disclosure, provides asurprisingly synergistic effect on bioavailability relative toabsorption enhancer alone, or chelating agent alone. Without intendingto be bound by theory, an oral pharmaceutical composition of the presentdisclosure is believed to overcome a series of different and unrelatednatural barriers to bioavailability. Various components of thepharmaceutical compositions act to overcome different barriers bymechanisms appropriate to each, and result in synergistic effects on thebioavailability of a Class II, III or IV drug. Some Class II, III or IVdrugs, taken alone or in the presence of salts of cationic metals, havereduced bioavailability due to chelate formation in the gut. Withoutintending to be bound by theory, it is believed that when a sufficientamount of citric acid is used as a chelating agent in a composition ofthe present disclosure, the citric acid can act as a high affinitychelating agent. As used herein, the term “high affinity chelatingagent” means a chelating agent that exhibits a low equilibriumdissociation constant, K_(d) towards the respective metal salt inquestion. For example, the citric acid is believed to chelate thecationic metal salts, which are therefore not available to interferewith the body's ability to absorb the Class II, III or IV drug. Withoutintending to be bound by theory, it appears that, in accordance with thepresent disclosure, a Class II, III or IV drug administered in apharmaceutical composition of the present disclosure is transportedthrough the stomach along with the chelator (i.e., the dosage form ispassed intact past the pylorus).

According to aspects illustrated herein, there is disclosed a method fortreating a patient having a gram-positive or a gram-negative bacterialpathogen comprising oral administering to the patient a solid dosageform, wherein the solid dosage form comprises a therapeuticallyeffective amount of tigecycline, at least one chelating agent and atleast one absorption enhancer. In an aspect, there is disclosed a methodfor treating a patient having complicated skin and skin structureinfections (cSSSI) comprising oral administering to the patient a soliddosage form, wherein the solid dosage form comprises a therapeuticallyeffective amount of tigecycline, at least one chelating agent and atleast one absorption enhancer. In an aspect, there is disclosed a methodfor treating a patient having complicated intra-abdominal infections(cIAI) comprising oral administering to the patient a solid dosage form,wherein the solid dosage form comprises a therapeutically effectiveamount of tigecycline, at least one chelating agent and at least oneabsorption enhancer. In an aspect, at least one of the chelating agentsis citric acid in a sufficient amount.

In accordance with the present disclosure, patients in need of treatmentwith Class II, III or IV drugs are provided with a pharmaceuticalcomposition thereof (at appropriate dosage), preferably but notnecessarily in tablet or capsule form of an ordinary size in thepharmaceutical industry. The dosages and frequency of administering theproducts are discussed in more detail below. Patients who may benefitare any who suffer from disorders that respond favorably to increasedlevels of a drug. For example, antibiotics in accordance with thepresent disclosure may be used to treat patients with bacterialinfection, protozoan infection and immunomodulation. In addition,antibiotics in accordance with the present disclosure may be used toprevent infection in a surgical wound, as a dental antibioticprophylaxis and for conditions of neutropenia. For example, vitamins inaccordance with the present disclosure may be used to treat patientswith a vitamin deficiency. Well-known human vitamin deficiencies involvethiamine (beriberi), niacin (pellagra), vitamin C (scurvy), and vitaminD (rickets).

By definition, Class II, III or IV drugs, taken alone, have reducedbioavailability due to precipitation in gastrointestinal media, theirphysicochemical properties preclude membrane permeation (either aninherent function of the molecule due to physicochemical properties, orthrough transport mechanisms, membrane deposition, protein-druginteractions, metabolism, etc.), or a combination thereof. With respectto a number of BCS class II, III or IV drugs, precipitation orinsolubility can be an inherent property of the molecule in question, orcan occur through ionic interactions, such as in the presence ofinorganic, or organic salts, hydrophobic interactions, etc. In anaspect, a carboxylic acid acts as a high affinity chelating agent of thepresent disclosure. Without intending to be bound by theory, it appearsthat, in accordance with the present disclosure, a Class II, III or IVdrug administered by a pharmaceutical composition of the presentdisclosure is transported through the gastrointestinal epithelium. Theacid is believed to chelate cationic metals, thereby inhibitingsalt-induced precipitation, while also acting as an enhancer ofparacellular absorption.

In an aspect, the acid is multifunctional and acts as a calciumchelator, a pH lowering agent, a bioavailability enhancer, a permeationenhancer and a membrane wetting/charge dispersal agent. By chelatingcalcium, the acid may act as a permeation enhancer by opening tightjunctions. Moreover, a chelator that is a pH lowering agent can furtherenhance paracellular permeation by inducing intracellular acidosis,resulting in actomyosin contraction through a PKC mediated event. Bysequestering free metals, it is no longer available to inhibit drugsolubility, or induce precipitation through complex formation, orsalt-induced precipitate formation, or maintain the tight junctionalintegrity through cadherin interactions. For example, different types ofantibiotics have been shown to interact with calcium. Further, citricacid can also act as a pH lowering agent, thereby enhancing paracellularabsorption through induction of intracellular acidosis, resulting inactomyosin contraction and increased paracellular flux.

Further, without being bound by theory, through its ability to modifymembrane charge dispersal, carboxylic acids can modify the action ofmembrane bound transporter proteins, thereby increasing absorptive flux.

The mechanism by which the present disclosure is believed to accomplishthe goal of enhanced bioavailability of the Class II, III or IV drug isaided by having active components of the pharmaceutical compositionreleased together as simultaneously as possible. The absorptionenhancer, which may be a solubility enhancer and/or transport enhancer(as described in more detail below), aids transport of the drug from thegastrointestinal tract to the blood. Many surface active agents may actas both solubility enhancers and transport (uptake) enhancers. Againwithout intending to be bound by theory, it is believed that enhancingsolubility provides (1) a more simultaneous release of the activecomponents of the present disclosure into the aqueous portion of theintestine, (2) better solubility of the drug in, and transport through,a mucous layer along the intestinal walls. Once the drug reaches theintestinal walls, an uptake enhancer provides better transport throughthe brush border membrane of the intestine into the blood, via eithertranscellular or paracellular transport. As discussed in more detailbelow, many preferred compounds may provide both functions. In thoseinstances, preferred aspects utilizing both of these functions may do soby adding only one additional compound to the pharmaceuticalcomposition. In other aspects, separate absorption enhancers may providethe two functions separately.

In an aspect, a single solid dosage form is used at each administration.Near simultaneous release is best achieved by administering allcomponents of the present invention as a single tablet, pill or capsule.However, the present invention also includes, for example, dividing therequired amount of acid and enhancers among two or more capsules whichmay be administered together such that they together provide thenecessary amount of all ingredients.

Example 1. Administration of Tigecyline in Rats Materials Animals andTest Article

Naïve, female Sprague-Dawley Rats (Taconic Farms, Germantown, N.Y.)housed in groups of three were maintained in a climate-controlled roomon a 12:12 h light-dark cycle with food and water available ad libitum.Animals weighed approximately 250 g at the time of testing. Rats werefasted overnight (with water available), prior to dosing. Information onthe test article, tigecycline, is listed in Table 1. Tigecycline stockwas prepared fresh on the day of each study in water and diluted in theindicated formulations.

TABLE 1 Test Article Information Compound Catalog Batch/Lot Item NameNumber Number Supplier Test Article Tigecycline S-1403 S140301 SelleckChemical Co.

Methods Doses and Route of Administration

Intravenous (IV) doses were administered as a bolus injection into theleft carotid artery at a dose volume of 1.6 mL/kg. Intraduodenal doseswere administered as bolus injections into the duodenum at a dose volumeof 1.2 mL/kg. Details of dosing and formulation composition for theprimary feasibility assessments are summarized in Table 2.

TABLE 2 Target Dosing and Formulations for Primary Feasibility StudiesDose Formulation Dose Concen- Composition Dose Volume tration CA³-SodiumTigecycline LLC² Citrate4 pH 3.5 Formulation Route mg/kg mL/kg mg/mL mMmM Study RA851 PBS¹ IV 0.6 1.6 0.4 — — A¹ ID 4.8 1.2 4.0 — — B ID 4.81.2 4.0 26 100 C ID 4.8 1.2 4.0 26 400 Study RA853 PBS¹ IV 12 1.6 7.5 —— A¹ ID 9.0 1.2 7.5 — — B ID 9.0 1.2 7.5 26 100 C ID 9.0 1.2 7.5 26 400¹Dulbecco's Phosphate Buffered Saline, Invitrogen (137 mM NaC1, 2.7 mMKC1, 10 mM Na2HPO4, 2 mM KH₂PO₄) ²Lauroyl-L-Carnitine, custom synthesis,Lonza ³Coated CA (DC F20), Jungbunzlauer 4Sodium citrate, dihydrate, J TBaker, Reagent

Additional studies were conducted comparing the % F and PK profiles oftigecycline formulated in one of each active excipient (RA861 andRA869), comparative to the highest % F formulation from the primaryfeasibility studies (400 mM CA and 26 mM LLC, pH 3.5, from studies RA851and RA853). An additional study was conducted exploring the effect offormulation pH at constant CA and LLC concentrations (RA867).

TABLE 3 Target Dosing and Formulations for Secondary Mechanistic StudiesDose Formulation Dose Concen- Composition Dose Volume tration CA³-SodiumTigecycline LLC² Citrate⁴ Formulation Route mg/kg mL/kg mg/mL mM mM⁵Study RA861 A ID 4.8 1.2 4.0 26 400 B ID 4.8 1.2 4.0 — 400 C ID 4.8 1.24.0 26 — Study RA867 A ID 4.8 1.2 4.0 26 400 B ID 4.8 1.2 4.0 26 400 (pH6.0) Study RA869 A¹ IV 0.6 1.6 0.4 — — B ID 4.8 1.2 4.0 26 400 C ID 4.81.2 4.0 26 — ¹Dulbecco's Phosphate Buffered Saline, Invitrogen (137 mMNaC1, 2.7 mM KC1, 10 mM Na₂HPO₄, 2 mM KH₂PO₄) ²Lauroyl-L-Carnitine,custom synthesis, Lonza ³Coated CA (DC F20), Jungbunzlauer ⁴Sodiumcitrate, dihydrate, J T Baker, Reagent ⁵Citrate buffer formulated at pH3.5 unless otherwise noted

Anesthesia and Catheterization

Female rats were maintained in groups of 3 per polycarbonate (orequivalent) shoebox cage with fresh wood chip bedding at 20-22° C. undera 12 hour on/off light:dark cycle. The rats were fasted overnight priorto surgery and anesthetized by intramuscular injection of 0.3 mL freshketamine (67 mg/mL)/xylazine (42 mg/mL) in 0.9% saline into the hindflank. After the rats went limp they were injected intraperitoneally(IP) with 0.1 mL ketamine/xylazine. The anesthetic state was maintainedby IP injection of ketamine/xylazine on an as needed basis. Animalweights were determined post-anesthesia induction.

An indwelling catheter for blood sampling was inserted into the rightcarotid artery by exposing the trachea with Mayo scissors, clamping thebottom of the artery and tying off the top with surgical suture. Thearea between was nicked with small sharp scissors and Intramedicpolyethylene tubing was inserted into the nicked area. The artery withthe tubing was sealed with suture to prevent leakage. A 23-gaugeIntramedic Luer Stub Adapter was inserted into the cannula and attachedto a 3 way valve which was connected to a 3 cc syringe filled with 0.9%normal saline and a 1 cc syringe filled with 5 U/mL sodium heparin forblood sampling.

IV Studies

In the IV phases, tigecycline was administered to groups of 3 naïveanesthetized female rats as a bolus injection away from the brain viathe left carotid artery that was constricted in the direction of thebrain. Blood samples (˜0.5 mL) for plasma tigecycline concentrationanalysis were collected from a surgically implanted cannula in the rightcarotid artery prior to dosing and at pre-determined time pointspost-dose up to either 120 or 240 minutes depending on the study. Bloodsamples were collected in tubes containing 20 μL 180 mM EDTA. The tubeswere maintained on ice and then centrifuged at approximately 3000 rpm at4° C. for 5 minutes. Plasma was harvested immediately aftercentrifugation of the samples and stored at −20° C. pending analysis.

Intraduodenal Studies

An intraduodenal injection was utilized to mimic oral delivery of anenterically coated capsule or tablet formulation. In the ID phase of thestudy, naïve female rats were administered tigecycline into the duodenumthrough a 1 mL syringe attached to a 27-gauge needle. Mayo scissors wereused to expose the duodenum and the site of administration wasidentified by measuring 5 cm from the junction of the stomach and theduodenum towards the jejunum. The measured site was identified byinserting a small piece of surgical suture underneath the site forinjection. After the formulation was injected, the abdominal cavity wasclosed with surgical clips. Blood samples (˜0.5 mL) were collected froma surgically implanted cannula in the right carotid artery for plasmatigecycline concentration analysis prior to dosing and at pre-determinedtime points post-dose up to either 120 or 240 minutes depending on thestudy.

Blood samples were collected in tubes containing 20 μL 180 mM EDTA. Thetubes were maintained on ice and then centrifuged at approximately 3000rpm at 4° C. for 5 minutes. Plasma was harvested immediately aftercentrifugation of the samples and stored at −20° C. pending analysis.

Analytical Procedure for Tigecycline

The quantitative determination of tigecycline in rat plasma wasperformed using an HPLC assay with UV detection at 350 nm. Minocycline(VWR international) was used as an internal standard. Sampleprocessing/clean-up of plasma samples was carried out offline by proteinprecipitation with acidified acetonitrile.

The HPLC system consisted of Shimadzu SIL-HTc HPLC system equipped withdual Shimadzu LC-10ADvp isopumps, a Shimadzu CTO-10ASvp columntemperature controller and a Shimadzu SPD-10Avp variable wavelengthdetector. The chromatographic separation was based on Li et al. withsome notable differences (See Li et al., 2004, Quantitation oftigecycline, a novel glycylcycline, by liquid chromatography. J.Chromatography B. 811:225-229). HPLC separation was achieved on areversed phase column (Phenomenex Luna C18(2), 5 μm, 150×4.6 mm, partnumber: 00F-4252-E0), using an initial isocratic phase, followed bygradient elution of tigecycline. Mobile phase A consisted of 23 mMphosphate buffer pH 2.5 with 6 mM 1-octanesulfonic acid, while mobilephase B consisting of pure acetonitrile. The time program startedisocratic at 25% mobile phase B for 6 minutes, followed by a linearincrease in mobile phase B to 35% over the next 10 minutes (16 minutes).The system was equilibrated to 25% mobile phase B for an additional 2minutes, resulting in a total runtime of 18 minutes per sample. Themobile phase flow rate was 1.2 mL/min. Detection was performed using theSPD-10Avp variable wavelength detector set at 350 nm, with a sensitivityof 0.001 aufs.

Both unknown samples and calibration standards (tigecycline in pooledrat plasma) were treated by protein precipitation with acetonitrilespiked with internal standard. The samples were then centrifuged underrefrigeration at 13 k rpm for 30 minutes. The supernatant was taken andthe liquid was removed to dryness in a turbovap. The samples were thenreconstituted in 55 μL 0.1M phosphate buffer, pH 3.8. Samples weremaintained at 10° C. while on the autoinjector during sequence. Theinjection volume was 50 μL. The unknown concentration in rat plasmasamples was determined by interpolation of the peak area ratios ofanalyte:internal standard versus the ratio of their nominalconcentrations into the regression line obtained from calibrationstandards spiked in pooled rat plasma. No regression weighting was usedfor the calculations. The method was demonstrated to be linear to 0.05μg/mL (defined LOQ). The calibration curve covered the range of 0.05μg/mL to 5.0 μg/mL.

Due to analytical issues observed in the analyses of earlier studysamples, the analytical method was changed slightly for later samples.Mobile phase A was changed to 23 mM phosphate buffer pH 2.5 with 4 mM1-octanesulfonic acid, while mobile phase B was changed to 90%acetonitrile, 10% water, with 2 mM 1-octanesulfonic acid. The timeprogram started isocratic at 25% mobile phase B for 6 minutes, followedby a linear increase in mobile phase B to 35% over the next 8 minutes(14 minutes), followed by re-equilibration to 25% mobile phase B for anadditional 4 minutes, resulting in a total runtime of 18 minutes. Thetotal runtime, detection wavelength, flow rate, injection volume, columnand column temperature all remained unchanged. Further, to assist insample cleanup, the protein precipitation reagent was acidified with0.5% v/v TFA. The resulting precipitate was centrifuged at 13 k rpm for5 minutes and the dried extract was reconstituted in 60 μL of mobilephase A. The reconstituted pellet was further centrifuged at 5 k rpm topellet any insoluble matter and the supernatant was injected. Thesechanges, presented side-by-side in Table 4 below, resulted in a morerobust analytical method, which exhibited better resolution betweentigecycline and minocycline, a better signal to noise ratio andeliminated the precipitation problems observed with earlier runs. Itshould be noted however, that when the method was transferred to anolder HPLC system (equipped with a Shimadzu SCT-10Avp system controllerand SIL-10A autoinjector), the time program was further altered suchthat the gradient went to 38% mobile phase B to account to system designchanges. All other parameters remained unchanged.

TABLE 4 RP-HPLC Analytical Methods Comparison for Tigecycline ParameterInitial Method Revised Method Column Phenomenex LUNA ™ C18(2) PhenomenexLUNA ™ C18(2) 5 μm, 150 × 4.6 mm 5 μm, 150 × 4.6 mm Part number:00F-4252-E0 Part number: 00F-4252-E0 Column Temperature 40° C. 40° C.Flow Rate 1.2 mL/min 1.2 mL/min Detection Wavelength 350 nm 350 nmInjection Volume 50 μL 50 μL Mobile Phase A 23 mM phosphate buffer pH2.5 23 mM phosphate buffer pH 2.5 6 mM 1-octanesulfonic acid 4 mM1-octanesulfonic acid Mobile Phase B 100% Acetonitrile 90% Acetonitrile(v/v) 10% Water (v/v) 2 mM 1-octanesulfonic acid Time Program 0-6 min:25% B 0-6 min: 25% B 6-16 min: linear to 35% B 6-14 min: linear to 35% B16-18 min: 25% B 14-18 min: 25% B Total Runtime 18 minutes 18 minutesStandard Curve 0.05-5.0 μg/mL 0.05-5.0 μg/mL Sample Preparation PPT:Acetonitrile PPT: 0.5% TFA (v/v) in Spin: 13k rpm 30 min AcetonitrileEvaporate to dryness Spin: 13k rpm 5 min Reconstitute in 55 μL 0.1MEvaporate to dryness phosphate buffer, pH 3.8 Reconstitute in 60 μL MP AInject Reconstitution Solution Spin: 5k rpm 5 min Inject SupernatantInternal Standard Prep 0.4 μg/mL in ppt solution 0.4 μg/mL in pptsolution (Minocycline)

Pharmacokinetic Data Handling

Tigecycline PK parameters for individual rats were calculated usingnon-compartmental analysis with PK Functions for Microsoft Excel.

Results: Primary Feasibility Studies (RA851, RA853) Plasma TigecyclineFollowing IV Administration

The mean C_(max) for plasma tigecycline at a target dose of 0.64 mg/kgwas 1.79 μg/mL and was observed at a mean time (T_(max)) of 5 minutes(0.08 hrs; Table 5. Tigecycline was measurable through 4 hours asexpected based on a reported single dose half-life of approximately 20hours. The mean AUC_((0-t)) was 58.20 μg*min/mL.

When the target dose was increased to 12 mg/kg (RA853), the mean C_(max)increased to 50.7 μg/mL, which was also observed at T_(max) of 5 minutes(Table 5). In both studies (RA851 and RA853), there is a clear biphasicdisposition of tigecycline, which is initially extremely fast, whichreaches a steady state by approximately 30 minutes (FIG. 1, FIG. 2, FIG.3). The mean AUC_((0-t)) in study RA853 was 791 μg*min/mL.

TABLE 5 Plasma Concentrations and Pharmacokinetics of Tigecycline inSprague-Dawley Rats Following a Single Dose IV Bolus Injection of 0.64mg/kg or 12 mg/kg Plasma Concentration (μg/mL) Target Dose Target Dose0.64 mg/kg (RA851) 12 mg/kg (RA853) Mean SEM Mean SEM Time (min) 0 0 0 00 5 1.790 0.268 50.691 11.954 10 0.888 0.152 26.194 6.615 20 0.447 0.0269.313 1.749 30 0.376 0.028 5.758 0.762 60 0.290 0.025 2.612 0.481 1200.216 0.108 1.866 0.359 240 0.116 0.116 Parameters C_(max) (μg/mL) 1.790.27 50.7 11.9 T_(max) (min) 5 0 5 0 AUC_((0-t)) 58.2 31.4 791 183 (μg ·min/mL) Animal Weight 0.238 0.018 0.217 0.012 (kg) Actual Dose 0.670.053 13.9 0.751 (mg/kg)

Plasma Tigecycline Following ID Administration

Tigecycline was administered by ID formulated in PBS, or in 26 mM LLCwith either 100 mM CA, pH 3.5, or 400 mM CA, pH 3.5. At either 4.8 or9.0 mg/kg target doses, tigecycline administered in PBS demonstratedlittle to no absorption (Table 6). In RA851 (4.8 mg/kg), the meanC_(max) was 0.10 μg/mL, which occurred at a mean T_(max) 90 minutes,while at the higher dose, the mean C_(max) was 0.22 μg/mL, occurring ata mean T_(max) of 30 minutes. This last result does not include oneanimal which demonstrated fairly significant absorption, with a C_(max)of 1.11 μg/mL at 10 minutes. The high degree of absorption, coupled withthe early T_(max) is uncharacteristic of the other 5 animals dosed withtigecycline formulated in PBS, indicating administration error throughinadvertent ID injection site leakage (resulting in IP administration).The mean % F based on AUC_((0-t)) when dosed at 4.8 mg/kg was 1.6%,comparative to the 0.64 mg/kg IV profile. When dosed at 9.0 mg/kg, themean % F was 2.8%.

In the presence of 26 mM LLC and 100 mM citrate, pH 3.5, the tigecyclineplasma concentrations increased significantly (7-9×), PK data aresummarized in Table 7. When dosed at 4.8 mg/kg (RA851), the meantigecycline C_(max) was 0.71 μg/mL, occurring at a mean T_(max) of 20minutes. The mean AUC_((0-t)) was 49.4 μg*min/mL, resulting in acalculated mean % F of 10.9% (CV 52.1% compared to 0.64 mg/kg IV).Increasing the dose to 9.0 mg/kg in the presence of 100 mM CA, 26 mM LLCalso showed a significant increase in tigecycline plasma concentrations,with a mean C_(max) of 2.06 μg/mL at mean T_(max) of 17 minutes. Themean AUC_((0-t)) was 76.9 μg*min/mL, resulting in a mean % F of 11.4%(CV, 59.3%). In short, the mean % F increased 4-7-fold when formulatedin 100 mM CA, pH 3.5, 26 mM LLC.

Increasing the citrate concentration to 400 mM, pH 3.5, in the presenceof 26 mM LLC resulted in additional increases in plasma tigecyclineconcentrations. Chromatograms demonstrate a secondarytigecycline-related peak with a longer retention time than the parenttigecycline peak, PK data are summarized in Table 8 and Table 9. Whendosed at 4.8 mg/kg, the mean C_(max) increased to 0.79 μg/mL (1.11 μg/mLwhen including the secondary peak) at a mean T_(max) of 17 minutes. Themean AUC_((0-t)) was 85.6 μg*min/mL (117.63 μg*min/mL including peak 2),resulting in a mean % F of 20.3% (CV, 96.6%; % F 27.9% including peak2).

Similar to the 100 mM CA experiments, increasing the dose to 9.0 mg/kgresulted in a significant increase in plasma tigecycline. The appearanceof the tigecycline-related peak was noted in this experiment as well,but it was not nearly as high comparative to the lower-dose experiment.When dosed at 9.0 mg/kg in 400 mM CA, pH 3.5, and 26 mM LLC, the meanC_(max) was 2.49 μg/mL (2.88[Lg/mL when including peak 2), occurring ata mean T_(max) of 10 minutes. The mean AUC_((0-t)) was 138 μg*min/mL(153 μg*min/mL including peak 2), resulting in a mean % F of 21.8% (CV,40.8%; % F 24.13% including peak 2). FIG. 3 compares the compiled meandose adjusted data from studies RA851 and RA853 for the differentformulations studied.

TABLE 6 Plasma Concentrations and Pharmacokinetics of Tigecycline inSprague-Dawley Rats Following a Single Dose ID Injection formulated inPBS Plasma Concentration (μg/mL) Target Dose Target Dose 4.8 mg/kg(RA851) 9.0 mg/kg (RA853) Mean SEM Mean* SEM Time (min) 0 0 0 0 0 100.031 0.031 0 0 20 0.031 0.031 0 0 30 0.033 0.033 0.391 0 60 0 0 0.440 0120 0.053 0.027 0.129 0 240 0.066 0.066 Parameters C_(max) (μg/mL) 0.100.06 0.22 0.22 T_(max) (min) 90 75.5 30 30 AUC_((0-t)) 6.96 6.04 15.7415.74 (μg · min/mL) Animal Weight 0.237 0.001 0.215 0.011 (kg) ActualDose 5.06 0.03 10.5 0.51 (mg/kg) % F^(†) 1.60 1.39 2.76 2.76 *Mean PKparameters do not include animal RA853-6, while mean reported plasmaconcentrations are representative of animal RA853-4 only due to death ofRA853-5. ^(†)% F for 4.8 mg/kg dose calculated relative to 0.64 mg/kg IVdata, while that of 9.0 mg/kg calculated relative to 12 mg/kg IV data asthose surgeries were performed on the same respective days.

TABLE 7 Plasma Concentrations and Pharmacokinetics of Tigecycline inSprague-Dawley Rats Following a Single Dose ID Injection formulated in100 mM CA (pH 3.5), 26 mM LLC Plasma Concentration (μg/mL) Target DoseTarget Dose 4.8 mg/kg (RA851) 9.0 mg/kg (RA853) Mean SEM Mean* SEM Time(min) 0 0 0 0 0 10 0.710 0.232 1.769 0.803 20 0.672 0.219 1.979 1.069 300.601 0.231 1.346 0.703 60 0.398 0.137 0.608 0.287 120 0.306 0.074 0.2730.185 240 Parameters C_(max) (μg/mL) 0.714 0.234 2.060 1.024 T_(max)(min) 20 5.774 16.667 3.333 AUC_((0-t)) 49.4 16.0 76.9 28.5 (μg ·min/mL) Animal Weight 0.231 0.006 0.193 0.006 (kg) Actual Dose 5.20 0.1311.7 0.38 (mg/kg) % F^(†) 10.85 3.26 11.36 3.89 ^(†)% F for 4.8 mg/kgdose calculated relative to 0.64 mg/kg IV data, while that of 9.0 mg/kgcalculated relative to 12 mg/kg IV data as those surgeries wereperformed on the same respective days.

TABLE 8 Plasma Concentrations and Pharmacokinetics of Tigecycline inSprague-Dawley Rats Following a Single Dose ID Injection formulated in400 mM CA (pH 3.5), 26 mM LLC (using parent Tigecycline peak only)Plasma Concentration (μg/mL) Target Dose Target Dose 4.8 mg/kg (RA851)9.0 mg/kg (RA853) Mean SEM Mean* SEM Time (min) 0 0 0 0 0 10 0.920 0.4682.492 0.405 20 0.788 0.337 2.160 0.525 30 0.618 0.291 1.764 0.489 600.455 0.231 1.061 0.287 120 0.345 0.151 0.683 0.139 240 0.386 0.176Parameters C_(max) (μg/mL) 0.792 0.335 2.492 0.405 T_(max) (min) 16.73.33 10 0 AUC_((0-t)) 85.6 47.3 137.6 33.6 (μg · min/mL) Animal Weight0.245 0.003 0.204 0.002 (kg) Actual Dose 4.90 0.05 11.0 0.13 (mg/kg) %F^(†) 20.30 11.3 21.8 5.13 ^(†)% F for 4.8 mg/kg dose calculatedrelative to 0.64 mg/kg IV data, while that of 9.0 mg/kg calculatedrelative to 12 mg/kg IV data as those surgeries were performed on thesame respective days.

TABLE 9 Plasma Concentrations and Pharmacokinetics of Tigecycline inSprague-Dawley Rats Following a Single Dose ID Injection formulated in400 mM CA (pH 3.5), 26 mM LLC (including Tigecycline-related Peak)Plasma Concentration (μg/mL) Target Dose 4.8 mg/kg Target Dose 9.0 mg/kgMean SEM Mean* SEM Time (min) 0 0 0 0 0 10 1.257 0.603 2.877 0.429 201.076 0.463 2.496 0.581 30 0.869 0.414 2.057 0.556 60 0.625 0.328 1.2220.371 120 0.485 0.225 0.782 0.191 240 0.511 0.200 Parameters C_(max)(μg/mL) 1.112 0.443 2.877 0.429 T_(max) (min) 16.7 3.33 10 0 AUC_((0-t))117.6 66.9 152.8 46.8 (μg · min/mL) Animal Weight 0.245 0.003 0.2040.002 (kg) Actual Dose 4.90 0.05 11.1 0.13 (mg/kg) % F^(†) 27.90 16.0124.13 7.22 ^(†)% F for 4.8 mg/kg dose calculated relative to 0.64 mg/kgIV data, while that of 9.0 mg/kg calculated relative to 12 mg/kg IV dataas those surgeries were performed on the same respective days.

Mechanistic Feasibility Assessments (RA861, RA867, RA869)

The positive data from the primary feasibility studies necessitatedinvestigation into the individual contributions of each active excipient(CA and LLC). Therefore, additional studies were conducted comparing the% F and PK profiles of tigecycline administered formulated in one ofeach of the active excipients (RA861 and RA869), comparative to thehighest % F formulation from the primary feasibility studies (400 mM CA,pH 3.5, and 26 mM LLC from studies RA851 and RA853). An additional studywas conducted exploring the effect of formulation pH at constant CA andLLC concentrations (RA867).

RA861 was designed with three ID injection arms, 400 mM CA, pH 3.5, with26 mM LLC as a control, 400 mM CA, pH 3.5, alone and a third armformulated with 26 mM LLC alone. Results demonstrated extremely lowexposure comparative to the primary feasibility studies, an issue whichwas believed to have stemmed from the analytical method. These issuesnecessitated study RA869, which was essentially a repeat of RA861, butreplacing the CA only arm with an IV comparator. In short, both studiesdemonstrate the utility of CA as an enabling excipient for tigecycline.While PK data from study RA869 replicated the approximately 22% % F whendosed in 400 mM CA, pH 3.5, and 26 mM LLC (RA851, RA853), dosing withonly 26 mM LLC resulted in a mean 9% F.

Study RA867 compared tigecycline % F and PK when dosed in formulationsat different pH. This study contained two formulation arms, where sixrats each were ID dosed with 400 mM CA and 26 mM LLC at either pH 3.5,or pH 6.0. Data demonstrate that rats dosed with formulation at pH 3.5demonstrate higher C_(max), earlier T_(max) and higher AUC_((0-t)).These data would suggest the import of pH, rather than calciumsequestration as the predominating mechanism underlying CA function asan enabling excipient for this small molecule.

TABLE 10 Summary of Mean Tigecycline Pharmacokinetic Parameters inFemale Sprague-Dawley Rats With Varying pH, CA and/or LLC Content (% CV)Mean Dose C_(max) T_(max) AUC_((0-t)) Study Study Arm (mg/kg) N (ng/mL)(min) (ng*min/mL) % F RA861 400 mM CA/ 4.6 4 30.6 (48.1) 10 (0) 8314(61.5) 2.1 (61.9) 26 mM LLC 400 mM CA 4.8 4 11.7 (125) 20 (173) 1050(195) 0.3 (195) 26 mM 2LLC 4.6 4 64.1 (62.9) 18 (54.7) 4082 (93.9) 1.0(92.3) RA867 400 mM CA/ 4.8 6 488 (48.0) 12 (35.0) 32202 (48.8) 7.7(47.1) 26 mM LLC pH 3.5 400 mM CA/ 4.8 6 278 (35.1) 43 (87.2) 23127(37.0) 5.5 (37.2) 26 mM LLC pH 6.0 RA869 IV 0.6 2 369 (39.9) 7.5 (47.1)7893 (23.3) — 400 mM CA/ 4.5 2 246 (17.9) 15 (47.1) 13339 (0.12) 21.6(5.47) 26 mM LLC 26 mM LLC 4.5 2 105 (47.0) 10 (0) 5500 (60.3) 8.9(59.9)

Primary Feasibility Studies (RA851, RA853)

These studies demonstrated the feasibility of improving the oral % F oftigecycline, a BCS Class III antibiotic, using the combination ofcitrate buffer (pH 3.5) and LLC in the anesthetized rat model.Intraduodenal administration was utilized to mimic administration of anenterically coated dosage form in larger mammals. Neither these primaryfeasibility studies, nor the mechanistic studies discussed below, weredesigned to explore whether or not enterically coating the dosage formis a necessity for small molecules. From a theoretical perspective,given the mechanism of permeation, enteric coating should be preferredfrom a variation point of view since it would limit potential food anddilution effects.

Intravenous administration of tigecycline resulted in an expectedbiphasic, first order plasma concentration curve. Given the variable andrelatively low tigecycline plasma concentrations observed in thedisposition phase of ID administered tigecycline in study RA851 (FIG. 7and FIG. 10), the studies were repeated at higher dose (RA853). From atheoretical perspective, one way to overcome low exposure upon dosing aBCS Class III compound is by increasing the local concentrationavailable for absorption (i.e., the dose). Commensurate with a passiveabsorption mechanism, while overall exposure was directly dependent onthe dose, the dose adjusted PK curves were virtually superimposable(FIGS. 3, 9, 12 and 15).

For small molecule therapeutics, which in general, do not require theacidifying activity of CA to inhibit metabolic enzymes, we hypothesizedthat CA would still be beneficial as an enabling excipient for certainBCS compounds. Indeed, the absolute tigecycline % F was also dependenton CA concentration, demonstrating its potential utility as both asolubilizing excipient (in this specific case only), as well as apermeation enhancer. Tetracycline analogues are known to interact withbile salts in the presence of calcium ions, resulting in complexprecipitation. Citric acid can disrupt these interactions by eitherchelating calcium, or by acidification of the milieu, thereby disruptingthese bile salt interactions and making the active available forabsorption. The absolute tigecycline % F was approximately 11% whenformulated with 100 mM CA, pH 3.5, and 26 mM LLC, which increased toapproximately 22% when formulated with 400 mM CA, pH 3.5, and 26 mM LLC(Table 11 and FIG. 16).

TABLE 11 Summary of Mean Tigecycline Pharmacokinetic Parameters inFemale Sprague-Dawley Rats (% CV) Mean Dose C_(max) T_(max) AUC_((0-t))Study Study Arm (mg/kg) N (μg/mL) (min) (μg*min/mL) % F* RA851 IV 0.7 31.79 (25.9) 5 (0) 58.2 (53.9) — PBS 5.1 3 0.10 (99.9) 90 (145) 6.96(150) 1.6 (150) 100 mM CA/ 5.2 3 0.71 (56.7) 20 (50) 49.4 (56.0) 10.9(52.1) 26 mM LLC 400 mM CA/ 4.9 3 0.79 (73.3) 17 (34.5) 85.6 (95.7) 20.3(96.6) 26 mM LLC RA853 IV 13.9 3 50.7 (40.8) 5 (0) 791 (40.1) — PBS 11.12 0.22 (141) 30 (141) 15.7 (141) 2.8 (141) 100 mM CA/ 11.7 3 2.06 (86.1)17 (34.6) 76.9 (64.3) 11.4 (59.3) 26 mM LLC 400 mM CA/ 11.0 3 2.49(28.1) 10 (0) 138 (42.3) 21.8 (40.8) 26 mM LLC *% F was calculatedcomparative to the respective IV arm of each study.

It should be stressed that the data for tigecycline formulated in 400 mMCA, pH 3.5, with 26 mM LLC presented in Table 11 are for tigecyclineonly. Upon analysis, it was noted that for both studies RA851 and RA853,chromatograms for plasma samples from this formulation exhibited afairly significant peak with a longer retention time than thetigecycline peak. This unknown peak (termed tigecycline-related peak)was not observed in the time zero plasma samples for the individualanimals, was observed in all subsequent time points and the levelcorresponded/followed the levels of tigecycline. It is hypothesized thatthis peak could potentially be a tigecycline degradation product,perhaps a citrate adduct, as there was some storage time between sampleacquisition and subsequent analysis, however the peak has not beenidentified. Further supporting the degradation theory is that this peakwas not observed in the mechanistic studies discussed below(specifically RA867 or RA869), where storage time was minimal. However,given that this peak has not been identified, it has not been includedin the overall summaries. Including the peak would result in an absolute% F of approximately 28% in RA851 and 24% in RA853 (Table 9).

Interestingly, when comparing the IV to the ID PK profiles, thedisposition phases of the curves are dramatically different (FIG. 20).Given the slower disposition (higher percent initial with respect totime), one can surmise that the bioavailabilities are underestimated bythe limited time of sample collection. Further studies would be requiredto determine why the disposition of ID administered tigecycline isslower than IV. One can suggest preferential tissue deposition, giventhe reported high steady state volume of distribution of tigecycline,but this is clearly speculation. It will be interesting to see if thisfinding is replicated in the dog model, especially consideringdifferences in hepatic and biliary anatomy and function in rat versusdog.

Mechanistic Feasibility Assessments (RA861, RA867, RA869)

The positive data from the primary feasibility studies necessitatedinvestigation into the individual contributions of each active excipient(CA and LLC). Therefore, additional studies were conducted comparing the% F and PK profiles of tigecycline formulated in one of each activeexcipient (RA861 and RA869), compared to the highest % F formulationfrom the primary feasibilities (400 mM CA, pH 3.5, and 26 mM LLC fromstudies RA851 and RA853). An additional study was conducted exploringthe effect of formulation pH at constant CA and LLC concentrations(RA867).

Studies designed to assess the relative individual contributions of CAand LLC (RA861 and RA869) demonstrate the utility of CA as an enablingexcipient for an oral formulation of tigecycline. Potential analyticalmethod issues aside, results from study RA869 replicate the relativelyhigh absolute % F (22%) when formulated in 400 mM CA, pH 3.5, and 26 mMLLC, but also show 9% absolute tigecycline % F when dosed in only 26 mMLLC (no CA; Table 10). These studies not only support the concomitantuse of CA and LLC together, but offer further avenues of study into theobserved synergism of the combination.

To this end, study RA867 was conducted to determine if the effects of CAwere indeed due to the physicochemical nature of citrate, or due to thelow pH. This study compared tigecycline % F and PK when dosed informulations of identical compositions (400 mM citrate, 26 mM LLC), ateither pH 3.5, or pH 6.0. Data demonstrate that rats dosed with theformulation at pH 3.5 demonstrate higher C_(max), earlier T_(max) andhigher AUC_((0-t)). These data would suggest the import of pH, ratherthan calcium sequestration as the predominating mechanism underlying thefunction of CA as an enabling excipient for this small molecule.Additional studies, which remove potential confounders, are required tofully elucidate the utility of acidic pH for this compound. For example,other studies to demonstrate the effects of milieu acidification onpermeability enhancement, which is a known permeation enhancementmechanism. In this instance, acidification would have the added benefitof disrupting tigecycline-bile salt complexation. While higher pHcitrate would be a better calcium chelator (reported pKa's at 3.1, 4.7and 6.4), which would also disrupt the tigecycline-bile saltinteractions and enhance permeation, it's unclear how effective calciumchelation would be in disrupting the bile salt interactions. In short,the observed difference in % F could simply be a function ofinsolubility.

Studies presented in Example 1 demonstrate an embodiment of a drug usedin a pharmaceutical composition of the present invention, whereinpharmaceutical composition is for oral delivery of the BCS Class IIIsmall molecule. The absolute % F of tigecycline delivered by IDinjection and formulated in PBS was 1.6% at low dose (RA851) and 2.8% athigh dose (RA853). When tigecycline was formulated in 100 mM CA, pH 3.5,and 26 mM LLC, the absolute % F increased to approximately 11%.Increasing the CA content to 400 mM, pH 3.5, with 26 mM LLC resulted inan increase in absolute % F to approximately 22%. Additional mechanisticstudies described in Example 1 demonstrated the synergism of the twofunctional excipients, as well as the importance of formulation pH inenabling oral % F of tigecycline.

Example 2. Administration of Zanamavir in Rats Materials Animals

Naïve, female Sprague-Dawley rats (Taconic Farms, Germantown, N.Y.) werehoused in groups of two with food and water available ad libitum.Animals were approximately 250 g at the time of testing. Rats werefasted overnight (with water available), prior to dosing.

Test Articles and Reference Substances

Information on the test article, zanamivir, is listed in Table 12.

TABLE 12 Test Article Information Compound Item Name Supplier Lot NumberTest Article zanamivir MedChem CS-0631-05684 Express

Vehicle

A stock solution of zanamivir was prepared by dissolving zanamivir in 2N HCl to a concentration of 40 mg/mL immediately before use. Aliquots ofthis stock solution were diluted into the indicated formulations.

Methods Formulation Preparation and Dosing

Rats (n=3) were dosed by IV via an in-dwelling catheter to the carotidartery with a volume of 400 μL at a dose of 0.16 mg zanamivir. Bloodsamples were collected from the carotid artery prior to dosing and at 5,10, 20, 30, and 60 min post-administration of the test article.

Rats (n=3 per formulation) were dosed via ID injection, 5 cm from thepyloric junction, with a bolus of 300 μL of each formulation (1.2 mgzanamivir). Blood samples were collected from the carotid artery priorto dosing and at 10, 20, 30, 60, 120, and up to 180 minpost-administration of the test article.

All blood samples were treated with 20 μL of 180 mM EDTA and thencentrifuged at 3000 rpm for 5 min. The plasma was collected, storedfrozen at −20° C., and then shipped to Absorption Systems on dry ice foranalysis.

TABLE 13 Formulations of Zanamivir Formulation Components IV¹ PBS¹ A² B²C² D² zanamivir (mg/mL)³ 0.4 4.0 4.0 4.0 4.0 4.0 Capmul MCM (% v/v) 0 070 0 0 0 propylene glycol (% v/v) 0 0 20 0 0 0 buffered citrate (mM), 00 0 100 400 0 pH 3.5⁴ LLC (% w/v)⁵ 0 0 0 1.0 1.0 1.0 ¹Solvent isphosphate buffered saline ²Solvent is deionized water ³Diluted from astock solution of 40 mg/mL zanamivir in 2N HCl ⁴Diluted from a 2M citricacid stock solution, pH 3.5 ⁵Diluted from a 20% w/v stock solution ofLLC in water

Animal Procedures for Zanamivir Plasma Concentrations

Plasma samples were analyzed by Absorption Systems using a qualifiedLC-MS/MS assay to determine the plasma concentrations of zanamivir.

Calculation of Bioavailability

The AUC of the plasma zanamivir concentration vs. time curves wascalculated using trapezoidal integration in Microsoft Excel. Absolutebioavailability was calculated according to the equation below:

${\% F_{{({0 - 1})}h}} = {\frac{{AUC}_{({0 - {1h}})}^{ID}}{{AUC}_{({0 - {1h}})}^{IV}} \times \frac{D^{IV}}{D^{ID}} \times 100}$

where D^(ID) is the ID dose (mg) divided by the weight (kg) of eachindividual rat, while D^(IV) is the IV dose (mg) divided by the meanbody weight (kg) of the rats in the IV arm.

Results

Individual plasma concentrations of zanamivir for the six formulationsinvestigated are listed below. The mean PK profiles are shown in FIG. 27and the PK data for each formulation are summarized in Table 14.

TABLE 14 Summary of PK Data by Formulation Formulation IV PBS A B C DAUC_((0-1 h)) 1183 (27.0) 3297 (27.2) 3518 (28.6) 3559 (75.6) 4726(29.8) 2302 (27.2) (ng*h/mL), Mean Value (% CV) % F_((0-1 h)), NA 41.6(28.4) 35.7 (25.0) 41.2 (70.3) 57.9 (22.6) 27.3 (26.6) Mean Values (%CV)

Because the stock solution of zanamivir was prepared in 2 N HCl in orderto solubilize the API, all of the vehicles were acidic. This resulted inenhanced absorption of zanamivir for all the formulations. However,relative to each other, the vehicle with the best bioavailability wasformulation “C”, containing 400 mM CA and 1.0% LLC.

TABLE 15 Plasma Concentrations (ng/mL) of Zanamivir: IV FormulationStudy RA885 Animal # (weight, kg) Time-point 1 2 3 (min) (0.213) (0.225)(0.223) Mean SD 0 0 0 0 0 0 5 2120 2650 2350 2373 266 10 1950 2260 15701927 346 20 1240 1660 1250 1383 240 30 327 1450 821 866 563 60 877 1140681 899 230

TABLE 16 Plasma Concentrations (ng/mL) of Zanamivir: Formulation A StudyRA885 Animal # (weight, kg) Time-point 4 5 6 (min) (0.205) (0.218)(0.214) Mean SD 0 0 0 0 0 0 10 5150 3140 3290 3860 1120 20 3660 33403090 3363 286 30 4350 3510 2190 3350 1089 60 5050 4280 2390 3907 1369120 NS 6360 3800 5080 NA

TABLE 17 Plasma Concentrations (ng/mL) of Zanamivir: Formulation PBSStudy RA886 Animal # (weight, kg) Time-point 1 2 3 (min) (0.242) (0.220)(0.233) Mean SD 0 0 0 0 0 0 10 3840 3960 3200 3667 409 20 3930 4380 25603623 948 30 4440 4670 2410 3840 1244 60 5090 4810 2380 4093 1490 1203480 3840 1810 3043 1083 180 3130 NS 2450 2790 NA

TABLE 18 Plasma Concentrations (ng/mL) of Zanamivir: Formulation B StudyRA886 Animal # (weight, kg) Time-point 4 5 6 (min) (0.237) (0.241)(0.219) Mean SD 0 0 0 0 0 0 10 1560 4260 10200 5340 4420 20 2540 18007920 4087 3340 30 3160 1050 7210 3807 3131 60 2580 1200 4860 2880 1848120 1780 1820 5460 3020 2113 180 2180 1560 NS 1870 NA

TABLE 19 Plasma Concentrations (ng/mL) of Zanamivir: Formulation C StudyRA886 (Animal # weight, kg) Time-point 7 8 9 (min) (0.223) (0.250)(0.255) Mean SD 0 0 0 0 0 0 10 12700 4690 6190 7860 4258 20 5970 45004650 5040 809 30 6470 3940 4880 5097 1279 60 4150 3020 3370 3513 578 1206660 2250 2890 3933 2383 180 NS 3420 NS 3420 NA

TABLE 20 Plasma Concentrations (ng/mL) of Zanamivir: Formulation D StudyRA886 Animal # (weight, kg) Time-point 10 11 12 (min) (0.233) (0.231)(0.233) Mean SD 0 0 0 0 0 0 10 3720 3600 3650 3657 60.3 20 1650 26901860 2067 550 30 2190 3360 2020 2523 730 60 1030 3410 1650 2030 1235 120898 3760 561 1740 1758 180 625 NS NS 625 NA

TABLE 21 Summary of Example 2 Formulations Tested Formulation ComponentsI.V.¹ PBS¹ A² B² C² D² zanamivir (mg/mL)³ 0.4 4.0 4.0 4.0 4.0 4.0 CapmulMCM (% v/v) 0 0 70 0 0 0 propylene glycol (% v/v) 0 0 20 0 0 0 bufferedcitrate (mM), 0 0 0 100 400 0 pH 3.5⁴ LLC (% w/v)⁵ 0 0 0 1.0 1.0 1.0¹Solvent is phosphate buffered saline ²Solvent is deionized water³Diluted from a stock solution of 40 mg/mL zanamivir in 2N HCl ⁴Dilutedfrom a 2M citric acid stock solution, pH 3.5 ⁵Diluted from a 20% w/vstock solution of LLC in water

Each of the five ID formulations was administered as a bolus injectiondirectly into the duodenum of rats at a dose of 1.2 mg (4.8 mg/kg),while the IV formulation was injected via an in-dwelling catheter to thecarotid artery at a dose of 0.16 mg (0.64 mg/kg). Blood samples werecollected from the carotid artery prior to dosing and at predeterminedtime-points post-dosing to determine plasma concentrations of zanamivirby an LC-MS/MS assay.

TABLE 22 Summary of Bioavailability by Formulation % F_((0-1 h)), MeanValues (% CV) PBS A B C D 41.6 35.7 41.2 57.9 27.3 (28.4) (25.0) (70.3)(22.6) (26.6)

Because the stock solution of zanamivir was prepared in 2 N HCl in orderto solubilize the API, all of the vehicles were acidified. This resultedin an enhancement of absorption of zanamivir for all the formulations.However, relative to each other, the vehicle with the bestbioavailability was formulation “C”, containing 400 mM CA and 1.0% LLC.

Example 3: Aminoglycoside Administration in Dogs Materials Animals

Adult Beagle dogs weighing approximately 10 to 15 kg were used in thestudy. The animals were housed at Sinclair Research Center, Columbia,Mo. either individually or in pairs in over-sized dog runs. Primaryenclosures were as specified in the USDA Animal Welfare Act (9 CFR Parts1, 2 and 3) and as described in the Guide for the Care and Use ofLaboratory Animals (National Academy Press, Washington, D.C., 1996). A12-hour light/12-hour dark photoperiod was maintained. Room temperaturewas set to be maintained at 20±5° C. Relative humidity was monitored butnot controlled. Animal room and pen cleaning were performed according tothe testing facility (Sinclair) SOPs.

TEKLAD® Certified Canine Diet was provided once daily in amounts (˜400grams) appropriate for the size and age of the animals. Tap water wasavailable ad libitum via automatic watering device or water bowls.

Animals were fasted overnight prior to drug administration andthroughout the blood collection period.

TABLE 23 Test Article Information Batch/Lot Test Item Number SupplierKanamycin 5023 Spectrum, Amresco Tobramycin 110M1191V Sigma Aldrich

Vehicle

For the IV study, kanamycin and tobramycin were prepared as concentratedsolutions in PBS and shipped frozen to Sinclair Research Center where itwas diluted in PBS and filter sterilized prior to administration. Fororal studies, kanamycin and tobramycin were blended with otherexcipients and transferred to capsules.

Methods Doses and Route of Administration

An IV dose of kanamycin was administered as a bolus injection into eachof three Beagle dogs at a dose of 0.1 mg/mL. An IV dose of tobramycinwas administered as a bolus injection into each of three Beagle dogs ata dose of 0.1 mg/mL. Details of formulation composition and dosing aresummarized in Table 24.

TABLE 24 IV Formulation and Dose Dose Dose No. Test Study ConcentrationVolume of Formulation Article No. (mg/mL) (mL) Dogs JSV-003-040Kanamycin SC434 0.1 1.0 3 JSV-003-044 Tobramycin SC435 0.1 1.0 3

Oral dosing of capsules was accomplished by administering the capsulesto the back of each dog's mouth. Kanamycin was delivered in eitherDRCAPS™ or VCAP PLUS™ capsules, both from Capsugel. The DRCAPS™ offeracid resistant properties and do not require coating. The VCAP PLUS™capsules are vegetarian HPMC capsules. Details of formulationcomposition and dosing for kanamycin capsules are summarized in Table25.

TABLE 25 Kanamycin Capsule Composition, Coating and Number of Dogs StudyKanamycin CA LLC PROSOLV ™ Capsule No. of Formulation No. (mg) (mg) (mg)(mg) Type Coated ¹ Dogs JSV-003-010 SC426 10 500 100 67 DRCAPS ™ no 4JSV-003-041 SC433 10 250 100 240 DRCAPS ™ no 5 JSV-003-005 SC426 10 500100 67 VCAP PLUS ™ yes 4 JSV-003-038 SC432 10 0 0 525 VCAP PLUS ™ no 6JSV-003-039 SC432 10 500 100 67 VCAP PLUS ™ no 6 JSV-003-052 SC438 10250 100 247 VCAP PLUS ™ yes 3 JSV-003-053 SC438 10 100 100 361 VCAPPLUS ™ yes 3 JSV-003-054 SC438 10 50 100 393 VCAP PLUS ™ yes 3 ¹ To 6%weight gain with Eudragit 7 L30-D55.

Tobramycin was delivered in enteric-coated VCAP PLUS™ capsules and thedetails of formulation composition and dosing are summarized in Table26.

TABLE 26 Tobramycin Capsule Composition, Coating and Number of DogsStudy Tobramycin CA LLC PROSOLV ™ Capsule No. of Formulation No. (mg)(mg) (mg) (mg) Type Coated ¹ Dogs JSV-003-050 SC436 10 0 0 525 VCAPPLUS ™ yes 8 JSV-003-051 SC436 10 500 100 67 VCAP PLUS ™ yes 8 ¹ To 6%weight gain with Eudragit 7 L30-D55.

Study Design and Plasma Sample Collection

Adult female Beagle dogs, weighing 9-15 kg were used in the oral and IVstudies. Dogs were fasted overnight before the beginning of each studybut were allowed free access to water. On the day of the study, onepre-dose blood sample of 3 mL was collected from each animal.Subsequently, each group of animals was given a single capsulecontaining either kanamycin or tobramycin blended in a specifiedformulation.

After administration of the drug, 3 mL blood samples were collected fromthe brachial vein at various time-points up to 240 minutes (4 hours)post-administration. Blood samples were collected into new heparinizedmonovette sampling syringes. The samples were placed on ice before beingcentrifuged for 10 minutes at approximately 2750 rpm at 2-8° C. Eachtube was labeled with the dog ID # and time-point, and they were storedat −20° C. pending shipment.

Analytical Procedure for Kanamycin and Tobramycin

Plasma kanamycin and tobramycin concentrations were determined with acompetitive ELISA kit from UC Biodevices (Fremont, Calif.) according to“ELISA for Detection of Kanamycin”. For determination of kanamycin bloodlevels, the kit kanamycin standards were used for the standard curve.For determination of tobramycin blood levels, tobramycin fromSigma-Aldrich was diluted and used for the standard curve. The standardcurve range for kanamycin was 0.2 ng/mL to 3.0 ng/mL. The standard curverange for tobramycin was 0.1 ng/mL to 5.0 ng/mL. Plasma samples werediluted appropriately with the kit assay buffer.

Kanamycin and Tobramycin ELISA Calculations

Mean plasma concentrations of kanamycin and tobramycin were calculatedusing a 5-parameter curve fit in SOFTMAX™ Pro software, Version 5.0.1(Molecular Devices).

Pharmacokinetic Data Handling

Kanamycin and tobramycin concentration-time data for each animal wereanalyzed by non-compartmental methods using PK functions for MicrosoftExcel. The maximum plasma concentration (C_(max)) values and their timesof occurrence (T_(max)) were obtained directly from the plasmaconcentration vs. time profiles. The areas under the plasmaconcentration-time curves (AUC_(last)) were estimated by the lineartrapezoidal rule from time zero to the time of the last observed plasmaconcentration.

Results

Individual plasma concentration of kanamycin and tobramycin at eachsampling time, as well as the corresponding PK parameter, are presentedin Table 27 through Table 38. Mean concentration time profiles forkanamycin and tobramycin after IV administration is shown in FIG. 28 andFIG. 29, respectively.

Plasma Kanamycin Following IV Administration

The C_(max) for plasma kanamycin following IV administration (SC434) was50 ng/mL and was observed at a mean time (T_(max)) of 5 minutes (Table27). The mean concentration profiles for kanamycin after IVadministration are shown in FIG. 28.

TABLE 27 Plasma Concentrations and Pharmacokinetics of Kanamycin inBeagle Dogs Following a Single Dose IV Bolus Injection Dog Number 52965297 5298 Mean SD % CV Kanamycin Plasma Concentration (ng/mL) Time (min)0 0 0 0 0 0 NA 5 77.73 40.56 3.97 40.75 36.88 91 10 70.40 37.75 7.5238.56 31.45 82 15 56.24 54.88 9.70 40.27 26.49 66 20 39.87 41.85 14.5932.10 15.20 47 30 43.81 42.52 17.48 34.60 14.84 43 40 29.91 34.08 17.5827.19 8.58 32 60 30.96 27.79 18.01 25.59 6.75 26 90 0 9.81 17.91 9.248.97 97 120 0 13.36 16.63 10.00 8.81 88 240 0 0 4.84 1.61 2.79 173Parameters Dose (mg) 0.10 0.10 0.10 0.10 C_(max) (ng/mL) 77.73 54.8818.01 50.21 30.13 60 T_(max) (min) 5 15 60 27 29 110 AUC (min*ng/mL)2787 3806 3169 3254 514 16

Plasma Tobramycin Following IV Administration

The C_(max) for plasma tobramycin following IV administration (SC435)was 31 ng/mL and observed at a mean T_(max) of 5 minutes (Table 28). Themean concentration profiles for tobramycin after IV administration areshown in FIG. 29.

TABLE 28 Plasma Concentrations and Pharmacokinetics of Tobramycin inBeagle Dogs Following a Single Dose IV Bolus Injection Dog Number 52995300 5301 Mean SD % CV Tobramycin Plasma Concentration (ng/mL) Time(min) 0 0 0 0 0 0 NA 5 38.41 31.97 23.29 31.22 7.59 24 10 34.59 30.4717.89 27.65 8.70 31 15 29.03 26.11 17.74 24.29 5.86 24 20 23.21 25.7815.71 21.57 5.23 24 30 20.31 22.23 15.58 19.37 3.42 18 40 15.89 20.257.60 14.58 6.43 44 60 11.01 15.45 6.52 10.99 4.47 41 90 5.83 13.10 3.727.55 4.92 65 120 6.09 6.26 2.49 4.95 2.13 43 240 0 0 0 0 0 NA ParametersDose (mg) 0.10 0.10 0.10 0.10 C_(max) (ng/mL) 38.41 31.97 23.29 31.227.59 24 T_(max) (min) 5 5 5 5 0  0 AUC (min*ng/mL) 1937 2331 1085 1784637 36

Plasma Kanamycin Following Oral Administration

The results for orally administered kanamycin in unformulated, uncoatedVCAP PLUS™ capsules (SC432) containing only PROSOLV™ (formulationJSV-003-038) are shown in Table 29. All dogs showed low levels ofbioavailability with a mean of 2.8%. Individual dog profiles are shownin FIG. 30.

TABLE 29 Plasma Concentrations and Pharmacokinetics of Kanamycin inBeagle Dogs Following a Single Oral Dose in PROSOLV ™ of UncoatedCapsules (Formulation JSV-003-038) Dog Number 5269 5270 5271 5272 52735274 Mean Plasma Concentration of Kanamycin (ng/mL) SD Time (min) 0 0 00 0 0 0 0 15 0 0 28.85 42.19 0 0 11.84 18.82 30 0 0 41.29 57.37 0 016.44 25.98 45 0 5.77 42.43 64.79 0 41.75 25.79 27.51 60 17.71 19.1446.83 66.07 57.00 55.55 43.72 20.52 75 29.24 28.29 51.73 55.37 65.1064.52 49.04 16.54 90 42.04 34.59 54.47 60.16 80.59 67.86 56.62 16.83 10537.34 42.76 46.18 44.25 91.84 76.02 56.40 22.10 120 34.01 57.00 54.1047.26 90.94 71.81 59.19 19.87 135 30.61 45.13 46.91 28.84 92.20 57.1050.13 23.19 150 30.62 42.34 50.50 34.72 103.40 67.80 54.90 27.18 16532.82 43.09 56.87 37.01 62.95 40.27 45.50 11.83 180 24.49 48.42 58.3533.32 47.60 35.45 41.27 12.34 195 24.40 48.39 44.96 18.11 49.01 37.3437.04 13.06 210 23.75 41.52 46.94 18.29 62.18 0.00 32.11 22.38 225 22.3944.58 33.32 11.69 0.00 25.20 22.86 15.71 240 18.14 36.62 32.92 12.2544.40 26.29 28.44 11.95 Parameters C_(max) (ng/mL) 42.04 57.00 58.3566.07 103.40 76.02 67.15 21.00 T_(max) (min) 90 120 180 60 150 105 11843 AUC (min*ng/mL) 5377 7790 10586 9067 12375 9807 9167 2408 F (%) 1.72.4 3.3 2.8 3.8 3.0 2.8 0.7

The addition of CA and LLC to the uncoated capsules resulted in asignificantly higher mean bioavailability of 12.4% (SC432). Theindividual dog results are shown in Table 30 Individual dog profiles areshown in FIG. 31.

TABLE 30 Plasma Concentrations and Pharmacokinetics of Kanamycin inBeagle Dogs Following a Single Oral Dose in Uncoated Capsules Formulatedwith CA and LLC (Formulation JSV-003- 039) Dog Number 5275 5276 52775278 5279 5280 Mean Plasma Concentration of Kanamycin (ng/mL) SD Time(min) 0 0 0 0 0 0 0 0 15 125.18 117.18 0 0 0 568.01 135.06 220.27 30113.44 476.63 0 540.77 30.91 451.15 268.82 246.33 45 97.42 524.01 429.31411.43 69.50 288.62 303.38 186.31 60 93.91 448.02 355.26 314.58 100.89236.67 258.22 142.01 75 93.17 398.77 328.84 263.23 149.84 246.59 246.74112.34 90 75.41 315.50 253.03 267.73 126.44 347.77 230.98 107.47 10560.57 276.76 225.11 295.92 107.98 278.55 207.48 99.46 120 70.25 254.06192.94 240.75 103.69 287.13 191.47 87.06 135 58.47 175.69 160.35 250.1293.71 319.33 176.28 96.82 150 54.76 137.56 149.16 170.54 90.54 221.72137.38 58.94 165 62.69 167.66 138.70 141.50 78.66 243.87 138.85 65.25180 54.56 133.03 116.16 157.23 84.22 224.03 128.21 59.25 195 41.53142.78 131.61 131.22 74.51 193.81 119.24 53.76 210 39.51 98.54 122.4184.51 92.27 168.90 101.02 42.90 225 23.88 0 99.89 112.16 58.57 127.6570.36 51.29 240 23.53 67.00 96.00 79.73 56.86 131.24 75.73 36.51Parameters C_(max) (ng/mL) 125.18 524.01 429.31 540.77 149.84 568.01389.52 200.86 T_(max) (min) 15 45 45 30 75 15 38 23 AUC (min*ng/mL)15209 54617 41262 51323 19352 59781 40257 18843 F (%) 4.7 16.8 12.7 15.85.9 18.4 12.4 5.8

Enteric-coated VCAP PLUS™ capsules containing 100 mg LLC were preparedwith varying concentrations of CA to test the effect of CA content onthe bioavailability of kanamycin. Formulation JSV-003-005 (SC426)contained 500 mg of CA and gave a mean bioavailability of 14.2%. Theindividual dog plasma results are shown in Table 31 and the individualdog absorption profiles are shown in FIG. 32.

TABLE 31 Plasma Concentrations and Pharmacokinetics of Kanamycin inBeagle Dogs Following a Single Oral Dose of Capsules Containing 500 mgCA (Formulation JSV-003-005) Dog Number 5216 5217 5218 5219 Mean PlasmaConcentration of Kanamycin (ng/mL) SD Time (min) 0 0 0 0 0 0 15 0 0 020.24 5.06 10.12 30 0 0 0 22.52 5.63 11.26 45 0 0 0 107.91 26.98 53.9660 20.40 138.68 0 229.76 97.21 107.45 75 248.33 340.76 0 341.61 232.68161.17 90 298.03 324.25 0 365.94 247.06 167.06 105 314.82 312.88 0255.86 220.89 149.78 120 313.20 284.70 504.25 274.29 344.11 108.02 135213.70 213.15 689.95 212.17 332.24 238.47 150 172.90 159.47 566.06250.95 287.35 190.14 165 193.60 147.50 458.86 121.18 230.28 155.29 180107.90 145.20 490.18 101.57 211.21 186.97 195 90.58 139.00 442.56 87.48189.91 170.08 210 61.89 124.80 289.09 84.07 139.96 102.78 225 69.09111.70 241.48 78.41 125.17 79.67 240 59.82 100.70 208.15 94.21 115.7264.18 255 45.01 97.00 180.94 91.60 103.64 56.57 270 41.85 65.50 123.2888.77 79.85 34.72 285 44.64 55.50 109.43 38.75 62.08 32.32 300 42.2749.00 86.88 34.67 53.21 23.20 Parameters C_(max) (ng/mL) 314.82 340.76689.95 365.94 427.87 175.96 T_(max) (min) 105 75 135 90 101 26 AUC(min*ng/mL) 34753 41779 65215 43118 46216 13186 F (%) 10.7 12.8 20.013.3 14.2 4.1

Formulation JSV-003-052 (SC438) contained 250 mg of CA and gave a meanbioavailability of 11.4%. The individual dog plasma results are shown inTable 32 and the individual dog absorption profiles are shown in FIG.33.

TABLE 32 Plasma Concentrations and Pharmacokinetics of Kanamycin inBeagle Dogs Following a Single Oral Dose of Capsules Containing 250 mgCA (Formulation JSV-003-052) Dog Number 5332 5333 5334 Mean PlasmaConcentration of Kanamycin (ng/mL) SD Time (min) 0 0 0 0 0 15 0 0 0 0 300 0 0 0 45 0 0 0 0 60 0 0 0 0 75 0 117.92 193.31 103.74 97.43 90 0230.56 362.97 197.84 183.68 105 0 525.55 323.03 282.86 265.07 120 0548.56 296.40 281.65 274.58 135 0 466.00 287.00 251.00 235.08 150 285.66477.72 212.42 325.27 137.01 165 312.68 371.57 223.29 302.51 74.66 180281.71 331.32 121.99 245.01 109.39 195 217.49 254.00 102.52 191.34 79.05210 150.35 182.29 95.36 142.67 43.97 225 53.33 155.56 76.96 95.28 53.52240 58.18 148.76 66.18 91.04 50.15 Parameters C_(max) 312.68 548.56362.97 408.07 124.24 (ng/mL) T_(max) 165 120 90 125 38 (min) AUC 1995556031 34925 36970 18125 (min*ng/mL) F (%) 6.1 17.2 10.7 11.4 5.6

Formulation JSV-003-053 (SC438) contained 100 mg of CA and gave a meanbioavailability of 12.4%. The individual dog plasma results are shown inTable 33 and the individual absorption profiles are shown in FIG. 34.

TABLE 33 Plasma Concentrations and Pharmacokinetics of Kanamycin inBeagle Dogs Following a Single Oral Dose of Capsules Containing 100 mgCA (Formulation JSV-003-053) Dog Number 5335 5336 5337 Mean PlasmaConcentration of Kanamycin (ng/mL) SD Time (min) 0 0 0 0 0 15 0 0 0 0 300 0 0 0 45 0 0 0 0 60 0 0 0 0 75 0 42.07 0 14.02 24.29 90 0 58.50 019.50 33.77 105 0 68.20 0 22.73 39.38 120 267.56 64.99 170.59 167.71101.32 135 506.08 53.04 539.93 366.35 271.86 150 600.77 106.61 743.62483.67 334.26 165 357.58 61.39 716.79 378.59 328.20 180 340.01 53.18759.07 384.09 355.00 195 280.92 0.00 531.86 270.93 266.07 210 224.470.00 448.64 224.37 224.32 225 227.25 0.00 542.74 256.66 272.56 240194.29 0.00 408.40 200.90 204.28 Parameters C_(max) 600.77 106.61 759.07488.82 340.33 (ng/mL) T_(max) 150 150 180 160 17 (min) AUC 43527 762069862 40336 31243 (min*ng/mL) F (%) 13.4 2.3 21.5 12.4 9.6

Formulation JSV-003-054 (SC438) contained 50 mg of CA. Only one out ofthree dogs dosed showed detectable blood levels for kanamycin with amean bioavailability of 2.6%. The individual dog plasma results areshown in Table 34 and individual plasma absorption profiles are shown inFIG. 35.

TABLE 34 Plasma Concentrations and Pharmacokinetics of Kanamycin inBeagle Dogs Following a Single Oral Dose of Capsules Containing 50 mg CA(Formulation JSV-003-054) Dog Number 5338 5339 5340 Mean PlasmaConcentration of Kanamycin (ng/mL) SD Time (min) 0 0 0 0 0 15 0 0 0 0 300 0 0 0 45 0 0 0 0 60 0 0 0 0 75 0 0 0 0 90 0 0 0 0 105 0 0 0 0 120 0 00 0 135 0 0 0 0 150 0 0 0 0 165 0 0 347.67 115.89 200.73 180 0 0 344.80114.93 199.07 195 0 0 440.51 146.84 254.33 210 0 0 271.48 90.49 156.74225 0 0 213.69 71.23 123.37 240 0 0 193.05 64.35 111.46 ParametersC_(max) 0 0 440.51 146.84 254.33 (ng/mL) T_(max) NA NA 195 195 (min) AUC0 0 25720 8573 14850 (min*ng/mL) F (%) 0 0 7.9 2.6 4.6

The use of DRCAPS™ instead of enteric-coated VCAP PLUS™ on thebioavailability of kanamycin was investigated. Two studies wereundertaken with DRCAPS™, SC426 and SC433, respectively. In the firststudy (formulation JSV-003-010) DRCAPS™ contained 500 mg of CA and 100mg LLC. The mean bioavailability was 9.2% and the individual plasmakanamycin concentrations are shown in Table 35. The individual plasmaabsorption profiles are shown in FIG. 36.

TABLE 35 Plasma Concentrations and Pharmacokinetics of Kanamycin inBeagle Dogs Following a Single Oral Dose of Capsules Containing 500 mgCA and 100 mg LLC in DRCAPS ™ (Formulation JSV-003-010) Dog Number 52125213 5214 5215 Mean Plasma Concentration of Kanamycin (ng/mL) SD Time(min) 0 0 0 0 0 0 15 0 0 0 0 0 30 136.50 0 0 264.04 100.14 126.81 45227.00 0 0 319.54 136.64 162.23 60 218.50 34.64 0 446.62 174.94 204.9475 217.80 66.31 26.46 449.11 189.92 191.45 90 204.20 83.07 39.36 343.42167.51 136.43 105 188.20 88.13 25.87 327.70 157.48 131.72 120 145.7699.26 29.69 302.01 144.18 115.53 135 186.50 85.78 29.11 299.80 150.30119.04 150 145.60 45.72 31.26 243.24 116.46 98.63 165 141.70 44.99 40.41162.11 97.30 63.63 180 136.90 34.39 42.75 140.19 88.56 57.84 195 118.8040.46 47.90 137.69 86.21 49.24 210 98.60 36.03 42.57 180.66 89.47 66.97225 86.60 25.30 43.66 94.69 62.56 33.45 240 70.40 35.39 56.18 103.4666.36 28.61 255 73.00 40.14 49.18 63.42 56.44 14.62 270 71.80 0.00 52.7760.42 46.25 31.81 285 68.20 0.00 56.41 53.28 44.47 30.34 300 58.30 0.0041.10 34.64 33.51 24.47 Parameters C_(max) (ng/mL) 227.00 99.26 56.41449.11 208 176 T_(max) (min) 45 120 285 75 131 107 AUC (min*ng/mL) 3847811394 9512 60131 29879 24122 F (%) 11.8 3.5 2.9 18.5 9.2 7.4

The second study with DRCAPS™ (formulation JSV-003-041) contained 250 mgof CA and 100 mg LLC. The mean bioavailability was 10.6% and theindividual plasma kanamycin concentrations are shown in Table 36. Theindividual plasma absorption profiles are shown in FIG. 37.

TABLE 36 Plasma Concentrations and Pharmacokinetics of Kanamycin inBeagle Dogs Following a Single Oral Dose of Capsules Containing 250 mgCA and 100 mg LLC in DRCAPS ™ (Formulation JSV-003-041) Dog Number 52815282 5283 5284 5285 Mean Plasma Concentration of Kanamycin (ng/mL) SDTime (min) 0 0 0 0 0 0 0 15 0 0 138.40 0 0 27.68 61.89 30 0 0 238.54 0 047.71 106.68 45 0 0 194.13 199.20 355.77 149.82 151.42 60 197.89 0159.40 374.52 306.43 207.65 144.17 75 711.54 76.02 152.98 299.22 274.63302.88 245.84 90 735.06 105.37 137.31 348.87 251.22 315.57 253.57 105686.58 115.45 133.58 186.53 175.35 259.50 240.53 120 547.75 109.89130.51 217.06 139.54 228.95 182.78 135 408.86 97.42 105.46 183.32 123.33183.68 130.30 150 285.29 97.00 88.12 181.08 113.70 153.04 82.43 165242.83 110.48 70.80 165.34 111.31 140.15 66.52 180 145.29 97.16 66.5866.68 102.74 95.69 32.41 195 149.45 77.36 59.94 87.04 70.94 88.95 35.23210 151.28 108.38 41.67 0.00 0.00 60.27 67.47 225 95.78 92.33 0.00 0.000.00 37.62 51.53 240 113.14 88.44 0.00 0.00 0.00 40.32 55.89 ParametersC_(max) (ng/mL) 735.06 115.45 238.54 374.52 355.77 363.87 232.05 T_(max)(min) 90 105 30 60 45 66 31 AUC (min*ng/mL) 66213 16966 24723 3463330374 34582 18879 F (%) 20.3 5.2 7.6 10.6 9.3 10.6 5.8

Plasma Tobramycin Following Oral Administration

The results for orally administered tobramycin in unformulated, VCAPPLUS™ capsules containing only PROSOLV™ (formulation JSV-003-050) areshown in Table 37. The mean bioavailability was 0.2%; only three out ofeight dogs showed detectable blood levels of tobramycin. Individualplasma profiles are shown in FIG. 38.

TABLE 37 Plasma Concentrations and Pharmacokinetics of Tobramycin inBeagle Dogs Following a Single Oral Dose in PROSOLV ™ (FormulationJSV-003-050) Dog Number 5308 5309 5310 5311 5312 5313 5314 5315 MeanPlasma Concentration of Kanamycin (ng/mL) SD Time (min) 0 0 0 0 0 0 0 00 0 15 0 0 0 0 0 0 0 0 0 30 0 0 0 0 0 0 0 0 0 45 0 0 0 0 0 0 0 0 0 60 00 0 0 0 5.41 0 2.81 1.03 2.03 75 0 0 0 0 0 9.94 0 0.00 1.24 3.51 90 0 00 0 0 9.24 0 4.45 1.71 3.42 105 0 0 0 0 0 8.07 0 5.45 1.69 3.21 120 0 00 0 0 7.99 3.05 3.12 1.77 2.87 135 0 0 0 0 0 4.97 3.99 5.42 1.80 2.51150 0 0 0 0 0 5.14 4.40 5.21 1.84 2.56 165 0 0 0 0 0 4.39 3.43 5.92 1.722.46 180 0 0 0 0 0 3.08 4.98 6.12 1.77 2.58 195 0 0 0 0 0 2.33 3.75 4.651.34 1.95 210 0 0 0 0 0 0 3.31 5.57 1.11 2.14 225 0 0 0 0 0 2.10 2.223.31 0.95 1.36 240 0 0 0 0 0 0 0 0 0 Parameters C_(max) (ng/mL) 0 0 0 00 9.94 4.98 6.12 2.63 3.89 T_(max) (min) NA NA NA NA NA 75 180 180 14561 AUC (min*ng/mL) 0 0 0 0 0 940 437 780 270 397 F (%) 0.0 0.0 0.0 0.00.0 0.5 0.2 0.4 0.2 0.2

With the addition of CA and LLC, the mean bioavailability for the 8 dogsincreased to 15%. All dogs given formulation JSV-003-051 showed bloodlevels of tobramycin. The PK results are shown in Table 38 and theindividual plasma profiles are shown in FIG. 39.

TABLE 38 Plasma Concentrations and Pharmacokinetics of Tobramycin inBeagle Dogs Following a Single Oral Dose with CA and LLC (FormulationJSV-003-051) Dog Number 5316 5317 5318 5319 5320 5321 5322 5323 MeanPlasma Concentration of Tobramycin (ng/mL) SD Time (min) 0 0 0 0 0 0 0 00 0 15 0 0 0 0 0 0 0 0 0 30 0 0 42.66 0 0 0 0 0 5.33 15.08 45 0 0 200.2860.76 0 0 0 111.78 46.60 74.57 60 0 0 217.26 247.28 50.93 0 0 168.5385.50 107.48 75 0 0 281.13 360.49 109.20 0 0 258.73 126.19 151.40 90 0 0264.55 328.17 174.06 0 0 266.34 129.14 144.17 105 0 0 253.75 411.29248.33 0 0 155.24 133.58 158.82 120 0 0 259.62 296.63 419.60 0 0 142.60139.81 167.09 135 14.99 0 180.86 277.83 235.47 65.64 0 108.01 110.35109.63 150 192.55 0 158.89 0.00 263.00 94.04 0 81.19 98.71 99.27 165238.77 0 143.39 244.02 187.52 90.04 81.32 64.12 131.15 87.44 180 294.340 126.93 200.20 175.01 86.65 384.60 59.47 165.90 126.62 195 256.81 0112.71 163.21 136.25 82.81 511.32 52.72 164.48 159.69 210 209.64 112.49101.78 155.59 118.59 71.71 503.29 22.30 161.92 148.53 225 169.12 204.4083.39 142.60 104.87 62.34 459.58 27.90 156.78 135.18 240 129.03 237.670.00 129.69 89.35 38.33 423.36 16.42 132.98 140.12 Parameters C_(max)(ng/mL) 294.34 237.67 281.13 411.29 419.60 94.04 511.32 266.34 314.47 12T_(max) (min) 180 240 75 105 120 150 195 90 144 57 AUC (min*ng/mL) 216116536 36408 44294 34013 8586 32277 22907 25829 13402 F (%) 12.1 3.7 20.424.8 19.1 4.8 18.1 12.8 14.5 7.5

Mean Oral Absorption Profiles of Kanamycin

A comparison for the mean absorption profiles for dogs administeredkanamycin in DRCAPS™ (JSV-003-010) and VCAP PLUS™ (JSV-003-005) capsulesare shown in FIG. 40. Both sets of capsules contained the same keyexcipients (500 mg CA and 100 mg LLC) and the only difference was thecapsules themselves. The data for each set of capsules were adjusted forthe mean T_(max). The error bars represent the standard error of themean (SEM).

A comparison for the mean absorption profiles for dogs administeredkanamycin in unformulated (0 mg CA and 0 mg LLC) VCAP PLUS™(JSV-003-038) and formulated (500 mg CA and 100 mg LLC) VCAP PLUS(JSV-003-039) capsules are shown in FIG. 41. Both sets of capsules wereuncoated. The data for each set of capsules were adjusted for the meanT_(max). The error bars represent the SEM.

A comparison for the mean absorption profiles for dogs administeredkanamycin in VCAP PLUS™ capsules with 100 mg of LLC and variousconcentrations of CA (500 mg CA, JSV-003-005; 250 mg CA, JSV-003-052;100 mg CA, JSV-003-053; 50 mg CA, JSV-003-054) are shown in FIG. 42. Thedata for each set of capsules were adjusted for the mean T_(max). Theerror bars represent the SEM.

Mean Oral Absorption Profiles of Tobramycin

A comparison for the mean absorption profiles for dogs administeredtobramycin in VCAP PLUS™ unformulated (0 mg CA and 0 mg LLC,JSV-003-050) capsules and formulated (500 mg CA and 100 mg LLC,JSV-003-051) are shown in FIG. 43. The data for each set of capsuleswere adjusted for the mean T_(max). The error bars represent the SEM.

DISCUSSION

Using a pharmaceutical composition of the present disclosure an oralbioavailability of 14% for kanamycin and 15% for tobramycin wasachieved. The key excipients are CA and LLC. In Example 3, aconcentration of at least 100 mg of CA is used in a pharmaceuticalcomposition. In the present example, the VCAP PLUS™ capsules arepreferable in a pharmaceutical composition to the DRCAPS™ capsules. Thestudies outlined in Example 3 serve as examples for BCS Class IIIaminoglycoside antibiotics. The examples of kanamycin and tobramycin arenot intended to be limiting and are used as examples that apharmaceutical composition of the present disclosure can improve oralbioavailability of BCS Class III molecules.

A feasibility study was carried out to evaluate the oral bioavailabilityand pharmacokinetics (PK) of the BCS Class 3 aminoglycoside bacterialantibiotics, kanamycin and tobramycin. The study design consisted of anintravenous (IV) phase and several oral phases dosed in Beagle dogs.

Orally administered kanamycin in an enteric-coated VCAP PLUS™ capsulecontaining 500 mg of citric acid (CA) and 100 mg lauroyl-L-carnitine(LLC) (formulation JSV-003-005) was given to 4 dogs and blood sampleswere collected up to 4 hours to determine plasma kanamycinconcentrations over time. The dogs showed a mean C_(max) of 428 ng/mLand a mean absolute bioavailability of 14%. Orally administeredtobramycin in an enteric-coated VCAP PLUS™ capsule containing the sameexcipients (formulation JSV-003-051) were given to 8 dogs and bloodsamples were collected up to 4 hours to determine plasma tobramycinconcentrations over time. These dogs showed a similar mean C_(max) of314 ng/mL and a similar mean absolute bioavailability of 15%.

TABLE 39 Summary of Mean Pharmacokinetic Parameters for Kanamycin andTobramycin Following Oral Administration of VCAP PLUS ™ Coated Capsulesto Beagle Dogs (±SEM) C_(max) T_(max) AUC_((0-t)) Formulation KeyExcipients N (ng/mL) (min) (ng*min/mL) % F JSV-003-005 10 mg Kanamycin,4 428 (88) 101 (13) 46216 (6593) 14.2 (2.0) 500 mg CA, 100 mg LLCJSV-003-051 10 mg Tobramycin, 8 314 (46) 144 (20) 25829 (4738) 14.5(2.7) 500 mg CA, 100 mg LLC

Pharmacokinetic studies with coated and uncoated capsules containingkanamycin formulated with various amounts of CA were studied in dogs.The oral bioavailability of kanamycin from uncoated capsules formulatedwithout CA and LLC was 2.8%, whereas the bioavailability of kanamycinfrom uncoated capsules formulated with 500 mg CA and 100 mg LLC was12.4%. There was no significant change in the bioavailability ofkanamycin orally delivered with enteric-coated capsules containing thesame formulation. Furthermore, there was essentially no difference inbioavailability when the CA levels were reduced to 100 mg. Decreasingthe amount of CA to 50 mg reduced the bioavailability of kanamycin to2.6%.

TABLE 40 Summary of Mean Pharmacokinetic Parameters for KanamycinFollowing Oral Administration of Vcap Plus (Coated and Uncoated)Capsules to Beagle Dogs (±SEM) C_(max) T_(max) AUC_((0-t)) FormulationKey Excipients N (ng/mL) (min) (ng*min/mL) % F JSV-003-038 0 mg CA, 6 67(9) 118 (18) 9167 (983)   2.8 (0.3) 0 mg LLC, uncoated JSV-003-039 500mg CA, 6 390 (82) 38 (9) 40257 (7693)  12.4 (2.4) 100 mg LLC, uncoatedJSV-003-005 500 mg CA, 4 428 (88) 101 (13) 46216 (6593)  14.2 (2.0) 100mg LLC, coated JSV-003-052 250 mg CA, 3 408 (72) 125 (22) 36970 (10465)11.4 (3.2) 100 mg LLC, coated JSV-003-053 100 mg CA, 3  489 (196) 160(10) 40336 (18038) 12.4 (5.5) 100 mg LLC, coated JSV-003-054 50 mg CA, 3¹  147 (147)  195 (NA) 8573 (8573)  2.6 (2.6) 100 mg LLC, coated ¹Onlyone dog out of three showed detectable blood levels.

Studies were also performed with DRCAPS™ containing 10 mg kanamycin, 100mg LLC and two concentrations of CA. These dogs did not show as high alevel of oral bioavailability. Results for these two studies arepresented below.

TABLE 41 Summary of Mean Pharmacokinetic Parameters for KanamycinFollowing Oral Administration of DR Capsules to Beagle Dogs (±SEM)C_(max) T_(max) AUC(0-t) Formulation Key Excipients N (ng/mL) (min)(ng*min/mL) % F JSV-003-010 500 mg CA, 4 208 (88)  131 (54) 29879(12061)  9.2 (4.0) 100 mg LLC JSV-003-041 250 mg CA, 5 364 (104)  66(14) 34582 (8443)  10.6 (2.6) 100 mg LLC

Studies with tobramycin comparing unformulated (0 mg CA and 0 mg LLC)and formulated (500 mg CA and 100 mg LLC) VCAP PLUS™ capsules aresummarized below. The oral bioavailability of tobramycin was 15% whichis similar to that of kanamycin, as expected based on similar molecularweight and structure.

TABLE 42 Summary of Mean Pharmacokinetic Parameters for TobramycinFollowing Oral Administration of Enteric-Coated VCAP PLUS ™ Capsules toBeagle Dogs (mean data (±SEM) are presented) C_(max) T_(max) AUC(0-t)Formulation Key Excipients N (ng/mL) (min) (ng*min/mL) % F JSV-003-050 0mg CA, 8¹ 3 (1) 145 (35) 270 (140) 0.15 (0.08) 0 mg LLC JSV-003-051 500mg CA, 8  314 (46)  144 (20) 25829 (4738)  14.5 (2.7)  100 mg LLC ¹Onlythree out of eight dogs responded with detectable blood levels.

Example 4: Tigecycline in Dogs Materials Animals and Test Article

Adult beagle dogs weighing approximately 9 to 15 kg were used in thestudy. The animals were housed at Sinclair Research Center, Columbia,either individually or in pairs in over-sized dog runs. Primaryenclosures were as specified in the USDA Animal Welfare Act (9 CFR Parts1, 2 and 3) and as described in the Guide for the Care and Use ofLaboratory Animals (National Academy Press, Washington, D.C., 1996). A12-hour light/12-hour dark photoperiod was maintained. Room temperaturewas set to be maintained between approximately 20±5° C. Relativehumidity was monitored but not controlled. Animal room and pen cleaningwere performed according to the testing facility (Sinclair) SOPs.

TEKLAD™ Certified Canine Diet was provided once daily in amounts (˜400grams) appropriate for the size and age of the animals. Tap water wasavailable ad libitum via automatic watering device or water bowls.Animals were fasted overnight prior to drug administration andthroughout the blood collection. Information on the test article,tigecycline, is listed in Table 43.

TABLE 43 Test Article Information Compound Catalog Batch/Lot Item NameNumber Number Supplier Test Article Tigecycline S-1403 S140301 SelleckChemical Co.

Methods Doses and Route of Administration

Doses are expressed in terms of free net tigecycline content.Intravenous doses of tigecycline were administered as bolus injectionsat a dose of 1.0 mg/mL. Oral dosing of enteric-coated capsules wasaccomplished by administering them to the back of the dog's mouth,followed by a chase of 5 mL water to ensure swallowing. Details ofdosing and formulation composition for the primary feasibilityassessments are summarized in Table 43.

TABLE 44 Capsule Composition, Dose and Number of Dogs ^(1, 2) CA³ LLC⁴PROSOLV ™ ⁵ Tigecycline Study (mg) (mg) (mg) (mg) Target DoseFormulation Number mg/capsule (mg/kg) n JSV-003-007 SC424 489 98 65 151.25 8 JSV-003-008 SC424 — — 482 14 1.25 8 JSV-003-032 SC430 465 93 4828 2.50 5 JSV-003-033 SC430 476 96 35 43 3.75 5 JSV-003-034 SC430 — —450 25 2.50 3 ¹ Capsule composition was calculated by multiplying theaverage powder content by the percentage of each component in the powderblend ² Target Dose assumes 12 kg average dog body weight at time ofdosing ³Citric acid DC F20, Jungbunzlauer, Unigene lot # AF659⁴Lauroyl-L-Carnitine, custom synthesis, Lonza, Unigene lot # AF946 ⁵PROSOLV ™ SMCC HD90, JRS Pharma, Unigene lot# AF602

Study Design and Sample Collection

Adult female Beagle dogs, weighing 9-15 kg were used in the oral and IVstudies. Dogs were fasted overnight before the beginning of each study.Water was provided ad libitum. On the day of the study, one pre-doseblood sample of 3 mL was collected from each animal. Subsequently, eachgroup of animals was dosed based on the protocol, i.e. either IV, or POby capsule.

After administration of the drug, 3 mL blood samples were collected fromthe brachial vein at various time-points up to 1140 minutes (24 hours)post-administration, depending on the study duration. Blood samples werecollected into new heparinized monovette sampling syringes. The sampleswere placed on ice before being centrifuged for 10 minutes atapproximately 2750 rpm at 2-8° C. Each tube was labeled with the dog ID# and time-point, and they were stored at −20° C. pending shipment.

Analytical Procedure for Tigecycline

The quantitative determination of tigecycline in dog plasma wasperformed using an HPLC assay using UV detection at 350 nm. Minocycline(VWR international) was used as an internal standard. Sampleprocessing/clean-up of plasma samples was carried out offline by proteinprecipitation with acidified acetonitrile using 0.5% trifluoroaceticacid (TFA).

The HPLC system consisted of Shimadzu SIL-HTc HPLC system equipped withdual Shimadzu LC-10ADvp isopumps, a Shimadzu CTO-10ASvp columntemperature controller and a Shimadzu SPD-10Avp variable wavelengthdetector. The chromatographic separation was based on Li et al. withsome notable differences (See Li et al., 2004, Quantitation oftigecycline, a novel glycylcycline, by liquid chromatography. J.Chromatography B. 811:225-229). HPLC separation was achieved on areversed phase column (Phenomenex Luna C18(2), μm, 150×4.6 mm, partnumber: 00F-4252-E0), using an initial isocratic phase, following bygradient elution of tigecycline. Mobile phase A consisted of 23 mMphosphate buffer pH 2.5 with 4 mM 1-octanesulfonic acid, while mobilephase B consisting of 90% acetonitrile, 10% water, with 2 mM1-octanesulfonic acid. The time program started isocratic at 25% mobilephase B for 6 minutes, followed by a linear increase in mobile phase Bto 35% over the next 8 minutes (14 minutes), followed byre-equilibration to 25% mobile phase B for an additional 4 minutes. Thetotal runtime was 18 minutes per sample. The mobile phase flow rate was1.2 mL/min. Detection was performed using the SPD-10Avp variablewavelength detector set at 350 nm, with a sensitivity of 0.001 aufs.

Both unknown samples and calibration standards (tigecycline in pooleddog plasma) were treated by protein precipitation with acidified (0.5%TFA v/v) acetonitrile spiked with internal standard. The samples werethen centrifuged under refrigeration at 13 k rpm for 5 minutes. Thesupernatant was taken and the liquid removed to dryness under nitrogenin a turbovap. The dried extract was reconstituted in 60 μL of mobilephase A and the suspension was further centrifuged at 5 k rpm for 5minutes to pellet any insoluble matter. The resulting supernatant wasthen injected onto the LC.

The unknown concentration in dog plasma samples was determined byinterpolation of the peak area ratios of analyte:internal standardversus the ratio of their nominal concentrations into the regressionline obtained from calibration standards spiked in pooled dog plasma. Noregression weighting was used for the calculations. The method wasdemonstrated to be linear to 0.05 μg/mL (defined LOQ). The calibrationcurve covered the range of 0.05 μg/mL to 5 μg/mL.

It should be noted however, that when the method was transferred to anolder HPLC system (equipped with a Shimadzu SCT-10Avp system controllerand SIL-10A autoinjector), the time program was further altered suchthat the gradient went to 38% mobile phase B to account to system designchanges. All other parameters remained unchanged.

TABLE 45 RP-HPLC Analytical Method for Tigecycline in Dog PlasmaParameter RP-HPLC Method Column Phenomenex LUNA ™ C18(2) 5 μm, 150 × 4.6mm Part number: 00F-4252-E0 Column Temperature 40° C. Flow Rate 1.2mL/min Detection Wavelength 350 nm Injection Volume 50 μL Mobile Phase A23 mM phosphate buffer pH 2.5 4 mM 1-octanesulfonic acid Mobile Phase B90% Acetonitrile (v/v) 10% Water (v/v) 2 mM 1-octanesulfonic acid TimeProgram 0-6 min: 25% B 6-14 min: linear to 35% B 14-18 min: 25% B TotalRuntime 18 minutes Standard Curve 0.05-5.0 μg/mL Sample Preparation PPT:0.5% TFA (v/v) in Acetonitrile Spin: 13k rpm 5 min Evaporate to drynessReconstitute in 60 μL MP A Spin: 5k rpm 5 min Inject SupernatantInternal Standard Prep 0.4 μg/mL in ppt solution (Minocycline)

Pharmacokinetic Data Handling

Tigecycline PK parameters for individual dogs were calculated usingnon-compartmental analysis with PK Functions for Microsoft Excel. Themaximum plasma concentration (C_(max)) values and their times ofoccurrence (T_(max)) were obtained directly from the plasmaconcentration vs. time profiles. The areas under the plasmaconcentration-time curves (AUC_(last)) were estimated with the lineartrapezoidal rule, by adding all the area portions under the curve fromtime zero to the time of the last observed plasma concentration.

Results Plasma Tigecycline Following IV Administration (SC427, SC431)

The mean C_(max) for plasma tigecycline at a target dose of 0.08 mg/kgwas 79.1 ng/mL and was observed at a mean time (T_(max)) of 5 minutes(0.08 hrs). Tigecycline was measurable through 4 hours as expected basedon a reported single dose half-life of approximately 20 hours in humans.The mean AUC_((0-t)) was 72.4 ng*hr/mL.

When the target dose was increased to 0.42 mg/kg, the mean C_(max)increased to 335 ng/mL, which was also observed at T_(max) of 5 minutes.In both studies, there is a clear biphasic disposition of tigecycline,which is initially extremely fast and reaches a steady state byapproximately 30 minutes. The mean AUC_((0-t)) was 411 ng*hr/mL.

TABLE 46 Summary of Tigecycline IV Pharmacokinetic Parameters in BeagleDogs Administered a Single 1 mg Dose Formulated in PBS (SC427) DogNumber 5220 5221 5222 Mean SD % CV Tigecycline Plasma Concentration(ng/mL) Time (min) 5 85.98 66.27 85.18 79.14 11.16 14.10 10 38.77 23.9233.00 31.90 7.48 23.46 15 26.49 19.85 20.60 22.32 3.64 16.29 20 27.7021.11 29.60 26.14 4.46 17.06 30 17.57 17.14 13.86 16.19 2.03 12.55 4014.30 11.51 21.58 15.80 5.20 32.89 60 13.52 12.49 14.50 13.51 1.00 7.4390 11.21 8.90 13.98 11.36 2.54 22.40 120 11.55 8.80 12.96 11.10 2.1219.06 360 0.00 0.00 3.45 1.15 1.99 173.21 1440 0.00 1.91 0.00 0.64 1.10173.21 Parameters Dose (mg/kg)* 0.08 0.08 0.08 — C_(max) (ng/mL) 85.9866.27 85.18 79.14 11.16 14.10 T_(max) (min) 5 5 5 5 0 0 AUC_((0-t))56.22 61.40 99.54 72.39 23.66 32.69 (ng*hr/mL) *Actual dog weights atthe beginning of studies were not available. Assumed 12 kg dog weight.

TABLE 47 Summary of Tigecycline IV Pharmacokinetic Parameters in BeagleDogs Administered a Single 5 mg Dose Formulated in PBS (SC431) DogNumber 5266 5267 5268 Mean SD % CV Tigecycline Plasma Concentration(ng/mL) Time (min) 5 335.66 250.27 419.13 335.02 84.43 25.20 10 197.55139.27 151.11 162.64 30.80 18.94 15 122.10 78.10 131.69 110.63 28.5825.83 20 117.20 67.21 98.92 94.44 25.30 26.79 30 102.81 49.42 76.3376.19 26.70 35.04 40 71.95 43.23 66.47 60.55 15.25 25.18 60 65.49 32.4649.22 49.06 16.52 33.67 90 59.88 30.78 51.58 47.41 14.99 31.61 120 53.2325.94 47.69 42.29 14.43 34.12 360 26.14 0.00 23.29 16.48 14.34 87.041440 0.00 0.00 0.00 0.00 0.00 0.00 Parameters Dose (mg/kg)* 0.42 0.420.42 — C_(max) (ng/mL) 335.66 250.27 419.13 335.02 84.43 25.20 T_(max)(min) 5 5 5 5 0 0 AUC_((0-t)) 568.95 153.68 509.94 410.86 224.67 54.68(ng*hr/mL) *Actual dog weights at the beginning of studies were notavailable. Assumed 12 kg dog weight.

TABLE 48 Summary of Mean Tigecycline IV Pharmacokinetic Parameters inBeagle Dogs (% CV) Dose C_(max) T_(max) AUC_((0-t)) Study (mg/kg) N(ng/mL) (min) (ng*hr/mL) Study 1 0.08 3 79.1 (14.1) 5 (0) 72.4 (32.7)(SC427) Study 2 0.42 3 335 (25.2) 5 (0)  411 (54.7) (SC431)

Plasma Tigecycline Following PO Administration (SC424, SC430)

Tigecycline was administered orally in enteric-coated capsules eitherformulated with 500 mg coated CA (Citrocoat DC F20, Jungbunzlauer), 100mg LLC, and silicified microcrystalline cellulose (PROSOLV™) as afiller, or unformulated with only filler. The capsules were entericallycoated using a standard preclinical protocol to 10% weight gain. Twostudies were conducted. In study SC424, tigecycline was administeredeither formulated or unformulated, both at a target 15 mg dose, to 8dogs each. Study SC430 consisted of dosing 30 mg tigecycline, bothformulated (n=5) and unformulated (n=3), and included an additional 45mg formulated arm (n=5) to study dose escalation affects.

Pharmacokinetic data demonstrate no exposure in either the 15 mg, or 30mg unformulated arms from studies SC424, or SC430, respectively, whileall formulated doses resulted in appreciable exposure in both studies.Oral dosing of 15 mg formulated tigecycline demonstrated a mean C_(max)of 75.5 ng/mL, a mean AUC_((0-t)) of 133 ng*hr/mL and a mean absolute %F of 12.2%. Pharmacokinetic results for individual dogs demonstrated arange of approximately 2 to 30% F. Tigecycline administration at higherdoses and with plasma sampling over 24 hours demonstrated increases inboth C_(max) and AUC_((0-t)) with dose. Dosing at 45 mg tigecyclineresulted in a mean C_(max) of 177 ng/mL, a mean AUC_((0-t)) of 574ng*hr/mL and a mean absolute % F of 15.5%. The mean T_(max) wasreproducible over all studied formulations.

TABLE 49 Summary of Tigecycline Pharmacokinetic Parameters in BeagleDogs Administered a Single PO 15 mg Unformulated, Enteric-Coated Capsule(SC424) Dog Number^(†) 5196 5197 5198 5199 5200 5201 Mean SD % CVTigecycline Plasma Concentration (ng/mL) Time (min) 15 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 30 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 45 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 60 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 75 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 90 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 105 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 120 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 135 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 150 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 165 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 180 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 195 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 210 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 225 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 2400.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Parameters Dose (mg/kg)*1.25 1.25 1.25 1.25 1.25 1.25 — C_(max) (ng/mL) 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 T_(max) (min) — — — — — — — — — AUC_((0-t))(ng*hr/mL) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % F 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 *Actual dog weights at the beginningof studies were not available. Assumed 12 kg dog weight. ^(†)Althoughthe initial study protocol indicated N = 8, only 6 dogs were analyzed.Dogs 5202 and 5203 were omitted from analysis.

TABLE 50 Summary of Tigecycline Pharmacokinetic Parameters in BeagleDogs Administered a Single PO 30 mg Unformulated, Enteric-Coated Capsule(SC430) Dog Number 5263 5264 5265 Mean SD % CV Tigecycline PlasmaConcentration (ng/mL) Time (hr) 0.0 0.00 0.00 0.00 0.00 0.00 0.00 0.30.00 0.00 0.00 0.00 0.00 0.00 0.7 0.00 0.00 0.00 0.00 0.00 0.00 1.0 0.000.00 0.00 0.00 0.00 0.00 1.3 0.00 0.00 0.00 0.00 0.00 0.00 1.7 0.00 0.000.00 0.00 0.00 0.00 2.0 0.00 0.00 0.00 0.00 0.00 0.00 2.3 0.00 0.00 0.000.00 0.00 0.00 2.7 0.00 0.00 0.00 0.00 0.00 0.00 3.0 0.00 0.00 0.00 0.000.00 0.00 3.3 0.00 0.00 0.00 0.00 0.00 0.00 3.7 0.00 0.00 0.00 0.00 0.000.00 4.0 0.00 0.00 0.00 0.00 0.00 0.00 4.5 0.00 0.00 0.00 0.00 0.00 0.005.0 0.00 0.00 0.00 0.00 0.00 0.00 5.5 0.00 0.00 0.00 0.00 0.00 0.00 6.00.00 0.00 0.00 0.00 0.00 0.00 8.0 0.00 0.00 0.00 0.00 0.00 0.00 10.00.00 0.00 0.00 0.00 0.00 0.00 24.0 0.00 0.00 0.00 0.00 0.00 0.00Parameters Dose (mg/kg)* 2.5 2.5 2.5 — C_(max) (ng/mL) 0.00 0.00 0.000.00 0.00 0.00 T_(max) (min) — — — — — — AUC_((0-t)) (ng*hr/mL) 0.000.00 0.00 0.00 0.00 0.00 % F 0.00 0.00 0.00 0.00 0.00 0.00 *Actual dogweights at the beginning of studies were not available. Assumed 12 kgdog weight.

TABLE 51 Summary of Tigecycline Pharmacokinetic Parameters in BeagleDogs Administered a Single 15 mg PO, Enteric-Coated Capsule Formulatedwith 500 mg CA and 100 mg LLC (SC424) Dog Number 5188 5189 5190 51915192 5193 5194 5195 Mean SD % CV Tigecycline Plasma Concentration(ng/mL) Time (min) 0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 15 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 30 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 45 0.00 0.00 67.620.00 0.00 0.00 0.00 0.00 8.45 23.91 282.84 60 0.00 0.00 102.56 0.00 0.000.00 46.91 0.00 18.68 37.66 201.55 75 67.41 0.00 124.71 0.00 0.00 44.9497.41 34.28 46.09 47.48 103.02 90 72.72 0.00 131.62 0.00 0.00 58.3674.02 93.83 53.82 49.47 91.92 105 72.29 0.00 114.31 94.07 12.21 59.0756.48 73.28 60.21 38.42 63.81 120 68.67 0.00 102.31 83.74 15.40 42.9247.02 51.68 51.47 33.73 65.54 135 58.13 0.00 97.81 75.90 12.41 39.1145.03 41.15 46.19 31.72 68.66 150 40.67 0.00 107.71 78.99 12.76 25.4435.51 32.23 41.66 35.29 84.70 165 44.69 10.79 90.92 76.85 12.42 24.2329.71 35.94 40.69 29.16 71.67 180 41.02 33.29 82.31 73.35 10.38 17.0621.14 20.67 37.40 26.80 71.65 195 37.51 38.70 82.52 75.43 6.06 14.6020.86 25.82 37.69 27.76 73.65 210 34.32 39.92 80.92 73.72 3.16 18.5119.45 20.23 36.28 27.69 76.31 225 35.89 33.39 80.91 69.32 0.00 12.7619.24 23.47 34.37 27.74 80.70 240 35.68 25.96 98.76 72.67 7.56 4.8916.45 19.58 35.19 33.40 94.89 Parameters Dose (mg/kg)* 1.25 1.25 1.251.25 1.25 1.25 1.25 1.25 — C_(max) (ng/mL) 72.72 39.92 131.62 94.0715.40 59.07 97.41 93.83 75.51 48.70 64.50 T_(max) (min) 90 210 90 105120 105 75 90 111 38.31 34.63 AUC_((0-t)) (ng*hr/mL) 148 42 329 184 2290 130 116 133 95.57 72.04 % F^(‡) 13.6 3.9 30.3 17.0 2.0 8.3 12.0 10.612.2 8.80 72.04 *Actual dog weights at the beginning of studies were notavailable. Assumed 12 kg dog weight. ^(‡)% F calculated from IV datagenerated from Study SC427.

TABLE 52 Summary of Tigecycline Pharmacokinetic Parameters in BeagleDogs Administered a Single 30 mg PO, Enteric-Coated Capsule Formulatedwith 500 mg CA and 100 mg LLC (SC430) Dog Number 5253 5254 5255 52565257 Mean SD % CV Tigecycline Plasma Concentration (ng/mL) Time (min)0.0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.3 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 0.7 0.00 95.94 0.00 0.00 36.01 26.39 41.89 158.74 1.00.00 154.19 0.00 0.00 90.62 48.96 70.71 144.42 1.3 0.00 182.24 0.00 0.0073.60 51.17 79.90 156.15 1.7 91.58 145.17 33.81 0.00 47.35 63.58 56.2088.38 2.0 66.04 127.57 87.39 NR 53.01 83.50 32.62 39.06 2.3 76.54 118.71120.08 0.00 45.11 72.09 51.04 70.81 2.7 55.33 107.93 87.68 0.00 44.3859.06 41.61 70.44 3.0 44.24 96.41 76.21 0.00 32.59 49.89 37.67 75.51 3.342.97 87.87 48.94 0.00 32.16 42.39 31.67 74.73 3.7 NR 71.18 50.58 0.00NR 40.59 36.63 90.24 4.0 33.78 65.95 48.33 0.00 35.01 36.61 24.23 66.194.5 34.53 61.09 38.99 0.00 33.07 33.54 21.88 65.24 5.0 33.82 63.28 34.270.00 27.47 31.77 22.53 70.93 5.5 NR 59.44 34.85 36.11 21.10 37.88 15.9041.99 6.0 0.00 NR 28.57 36.53 19.76 21.21 15.71 74.08 8.0 0.00 0.0018.56 0.00 17.77 7.27 9.95 136.99 10.0 0.00 0.00 0.00 0.00 12.91 2.585.77 223.61 24.0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Parameters^(†)Dose (mg/kg)* 2.5 2.5 2.5 2.5 2.5 — C_(max) (ng/mL) 91.58 182.24 120.0836.53 90.62 121.13 42.97 35.47 T_(max) (min) 1.7 1.3 2.3 6.0 1.0 1.60.57 35.95 AUC_((0-t)) (ng*hr/mL) 195 575 315 64 382 367 158.68 43.28 %F^(‡) 7.9 23.3 12.8 2.6 15.5 14.9 6.44 43.28 *Actual dog weights at thebeginning of studies were not available. Assumed 12 kg dog weight.^(†)Results reported excluding animal 5256 ^(‡)% F calculated from IV PKdata generated under Study SC431

TABLE 53 Summary of Tigecycline Pharmacokinetic Parameters in BeagleDogs Administered a Single 45 mg PO, Enteric-Coated Capsule Formulatedwith 500 mg CA and 100 mg LLC (SC430) Dog Number 5258 5259 5260 52615262 Mean SD % CV Tigecycline Plasma Concentration (ng/mL) Time (min)0.0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.3 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 0.7 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.0 133.63127.65 0.00 20.73 0.00 56.40 68.33 121.15 1.3 153.06 78.69 0.00 59.870.00 58.32 63.63 109.10 1.7 138.33 46.66 0.00 157.62 163.14 101.15 73.5172.67 2.0 116.10 43.10 44.20 149.31 367.01 143.94 132.93 92.35 2.3 87.3239.82 79.71 114.76 216.56 107.63 66.53 61.82 2.7 78.26 33.22 79.21102.49 199.64 98.56 61.82 62.73 3.0 74.06 31.63 62.12 98.25 116.52 76.5232.79 42.85 3.3 69.50 33.98 49.17 63.91 126.80 68.67 35.31 51.41 3.762.36 33.14 38.96 55.89 119.77 62.02 34.42 55.49 4.0 54.31 21.63 32.1446.68 103.81 51.71 31.76 61.41 4.5 58.85 16.87 21.77 47.61 99.47 48.9133.24 67.96 5.0 48.38 20.56 33.56 45.61 104.91 50.60 32.30 63.82 5.544.74 19.45 21.25 50.62 76.57 42.53 23.53 55.33 6.0 36.76 13.66 13.3834.52 77.87 35.24 26.29 74.59 8.0 27.89 10.52 6.37 20.09 49.82 22.9417.20 75.00 10.0 22.19 0.00 4.77 12.09 44.75 16.76 17.75 105.88 24.00.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Parameters Dose (mg/kg)* 3.753.75 3.75 3.75 3.75 — C_(max) (ng/mL) 153.06 127.65 79.71 157.62 367.01177.01 110.63 62.50 T_(max) (min) 1.33 1.00 2.33 1.67 2.00 1.67 0.5331.62 AUC_((0-t)) (ng*hr/mL) 682 232 237 546 1175 574 389 67.70 % F^(‡)18.4 6.3 6.4 14.8 31.8 15.5 10.52 67.70 *Actual dog weights at thebeginning of studies were not available. Assumed 12 kg dog weight. ^(‡)%F calculated from IV PK data generated under Study SC431

TABLE 54 Mean Tigecycline Pharmacokinetic Parameters Following OralAdministration in Enteric Coated Capsules (% CV). C_(max) T_(max)AUC_((0-t)) Study Formulation† N (ng/mL) (min) (ng*hr/mL) % F^(‡) Study1 15 mg Formulated 8  75.5 (48.7) 111 (38.3) 133 (72.0) 12.2 (72.0)(SC424) 15 mg Unformulated 6 ND — — 0.0 Study 2 30 mg Unformulated 3 ND— — 0.0 (SC430) 30 mg Formulated*  4*  121 (35.5)*  95 (36.0)*  367(43.3)*  14.9 (43.3)* 45 mg Formulated 5 177 (62.5) 100 (31.6) 574(67.7) 15.5 (67.7) *Results reported excluding animal 5256 †Formulatedcapsules targeted 500 mg CA and 100 mg LLC ^(‡)% F calculated based onthat study's respective IV arm

DISCUSSION

These studies demonstrated the feasibility of improving the oralbioavailability of tigecycline, a BCS Class III antibiotic, using thecombination of CA and LLC in beagle dogs. These formulations weredelivered in enterically coated capsules and provide a valuablepreclinical proof of concept to support potential future clinicaldevelopment. Example 1 conducted in rats using an ID injection modeldemonstrates the seemingly synergistic role of the combination of CA andLLC as absorption enhancing excipients. The studies outlined in Example1 also demonstrated the importance of formulation pH in enabling oral %F of tigecycline. While valuable from a mechanistic standpoint, Example1 used a liquid vehicle to directly deliver tigecycline to theabsorptive surface and was designed specifically to remove the potentialconfounder of the solid to liquid transition (disintegration anddissolution) of a solid dosage form, a primary factor in enablingbioavailability and controlling the variability inherent to enablingformulations.

Intravenous administration of tigecycline resulted in an expectedbiphasic, first order plasma concentration curve. Given that one way toovercome low exposure upon dosing a BCS Class III compound is byincreasing the local concentration available for absorption (i.e., thedose), the IV PK profile was determined at two concentrations. Whileoverall exposure was directly dependent on the dose (FIG. 44 and FIG.45), the dose adjusted PK curves were virtually identical (FIG. 46).

Rat studies (Example 1) also demonstrated the utility of higherconcentrations of CA in enabling higher tigecycline exposure. In thosestudies, 400 mM CA, pH 3.5, in combination with 26 mM LLC resulted inapproximately 21% bioavailability. It was hypothesized that CA could actto disrupt potential tetracycline:bile salt interactions, which lead totigecycline precipitation. To this end, these formulations were preparedwith a target of 500 mg coated CA, which is on the high end of theconcentration range typically studied, both clinically andpreclinically. Moreover, these studies did not explore the effects oflower CA concentrations, as data from the rat studies would immediatelysuggest lower net exposure. These studies did however explore theeffects of dose escalation on absolute exposure.

Peroral dosing of 15 mg (target 1.25 mg/kg) tigecycline with 500 mg CAand 100 mg LLC resulted in a mean C_(max) of 75.5 ng/mL, a meanAUC_((0-t)) of 133 ng*hr/mL and a mean absolute % F of 12.2%. Dosing at45 mg tigecycline resulted in a mean C_(max) of 177 ng/mL, a meanAUC_((0-t)) of 574 ng*hr/mL and a mean absolute bioavailability of15.5%. The mean T_(max), at approximately 100 minutes, was reproducibleover all studied oral formulations. Interestingly, while exposure waslinear with respect to both C_(max) and AUC, it was less than doseproportional with respect to both C_(max) and AUC_((0-4HR)) (Table 55).These observations could be a function of a limited absorption window(by design), as the percent difference from predicted (based on resultsfrom the 15 mg dose) at the 30 mg dose was lower than that for the 45 mgdose. An alternate hypothesis could be that other factors, such as bilesalt or transporter interactions, are playing a role in inhibitingabsorptive flux; i.e. as the dose is increased, tigecycline is in suchexcess that there isn't sufficient CA to inhibit either transportereffects, or precipitation with bile salt complexes. More likely,however, is that the biopharmaceutical properties of the molecule itselfresult in an observable saturation effect due to extensive tissuedistribution.

Tigecycline does indeed exhibit a high tissue distribution. Coupled witha lack of metabolism, the low dose proportionality further suggests bothinsufficient absorption, as well as nonlinear clearance with respect todose. In short, the dose relationship observed is a saturation effect,i.e. clearance decreases with respect to increasing dose, most likelydue to tissue deposition commensurate with the high tissue distributionof tigecycline, which could be confounded by absorption issues at thehigher doses. Further supporting this hypothesis is that no observabledifferences could be gleaned from the dose adjusted mean plasma profiles(FIG. 54). A relatively simple way to test this hypothesis would be tostudy the dose proportionality after single oral doses of tigecyclinefollowing a saturable IV infusion. Such a study would also benefitpotential further clinical investigation as this could be foreseen as apotential product profile for orally administered tigecycline.

TABLE 55 Dose Proportionality of Tigecycline Pharmacokinetic ParametersFollowing Oral Administration in Enterically Coated Capsules‡ PredictedPredicted Target Observed Mean C_(max) Observed AUC_((0-t)) Dose MeanC_(max) (ng/mL; % AUC_((0-t)) (ng*hr/mL; % (mg) (ng/mL) Difference)^(†)(ng*hr/mL) Difference)^(†) 15   75.5 — 133 — 30  121* 151 (−24.8)  214*265 (−23.9) 45 177 226 (−28.0) 266 398 (−49.4) *Results reportedexcluding animal 5256 ^(†)Predicted values based on 15 mg results^(‡)Note the time scale has been adjusted for comparison as SC424sampled to 4 hours, while SC430 sampled to 24 hours.

These studies demonstrate the feasibility of using the oral deliverytechnology for the BCS Class III small molecule tigecycline in the dogpreclinical model. No tigecycline exposure was observed in dogs dosedwith either 15 mg or 30 mg tigecycline capsules formulated in amicrocrystalline cellulose filler. Administration of enterically coatedcapsules containing 15 mg tigecycline and formulated with 500 mg CA and100 mg LLC resulted in a mean absolute % F of 12.2%. Increasing the doselinearly increased both C_(max) and AUC_((0-t)), but exposure was notdose proportional. The mean bioavailability of 45 mg tigecyclineformulated with 500 mg citric acid and 100 mg LLC resulted in a meanabsolute % F of 15.5%.

The purpose of these studies was to investigate the oral bioavailability(% F) and pharmacokinetic (PK) profiles of orally administeredtigecycline in beagle dogs. Two studies were conducted, each consistingof an intravenous (IV) phase and an oral phase with enteric-coatedcapsules containing formulated, or unformulated tigecycline. The studiesdiffered in the doses administered and sampling timeframe.

In Study 1 (SC424 and SC427), tigecycline was administered as a 1 mg IVbolus injection to beagle dogs (n=3), as well as a 15 mg, enteric coatedcapsule arm. The capsule phase included 2 formulations, eitherformulated with citric acid (CA) and 3,0-lauroyl-L-carnitine (LLC), orunformulated (n=8 dogs each). Venous blood samples were collected up to4 hours after dosing to determine plasma tigecycline concentrations withrespect to time.

Study 2 (SC430 and SC431) was designed based on feedback from Study 1,and included a higher dose IV arm (5 mg per animal, n=3), as well as 30mg and 45 mg formulated (n=5 each), enterically coated capsules and 30mg unformulated (n=3), enterically coated capsules. The study alsoincluded 24 hours venous sampling to fully characterize the PK profilesupon single oral doses. Formulated arms in both studies included 500 mgCA and 100 mg LLC, while unformulated arms included the drug dispersedin a microcrystalline cellulose filler. These parameters were not variedbetween Study 1 and Study 2.

Pharmacokinetic data indicate that IV administered tigecycline exhibiteda biphasic clearance, with an approximately proportional increase inboth C_(max) and AUC_((0-t)) upon increasing dose between Study 1 andStudy 2 (1 mg vs. 5 mg). The mean C_(max) was 79 ng/mL and 335 ng/mL,respectively, and the mean AUC_((0-t)) was 72.4 ng*hr/mL and 411ng*hr/mL, respectively.

TABLE 56 Summary of Mean Tigecycline IV Pharmacokinetic Parameters inBeagle Dogs (% CV) Dose C_(max) T_(max) AUC_((0-t)) Study (mg) N (ng/mL)(min) (ng*hr/mL) Study 1 1 3 79.1 (14.1) 5 (0) 72.4 (32.7) (SC427) Study2 5 3  335 (25.2) 5 (0)  411 (54.7) (SC431)

Oral administration of tigecycline formulated with microcrystallinecellulose (unformulated) and filled in enterically coated capsules ateither 15 mg (Study 1), or 30 mg (Study 2) did not result in observableplasma exposure, while tigecycline formulated with 500 mg CA and 100 mgLLC demonstrated appreciable exposure at all doses in both studies.Orally administered tigecycline formulated at 15 mg with activeexcipients were dosed to 8 dogs, with plasma sampling occurring over 4hours post-dose. Results demonstrate a mean C_(max) of 75.5 ng/mL, amean AUC_((0-t)) of 133 ng*hr/mL and a mean absolute % F of 12.2%.Tigecycline administration at higher doses and with plasma sampling over24 hours demonstrated increases in both C_(max) and AUC_((0-t)) withdose, but exposure was less than dose proportional, potentially due to alimited absorption window by design. Dosing at 45 mg tigecyclineresulted in a mean C_(max) of 177 ng/mL, a mean AUC_((0-t)) of 574ng*hr/mL and a mean absolute % F of 15.5%. The mean T_(max) wasreproducible over all studied doses.

TABLE 57 Mean Tigecycline Pharmacokinetic Parameters Following OralAdministration in Enteric Coated Capsules (% CV) C_(max) T_(max)AUC_((0-t)) Study Formulation ^(†) N (ng/mL) (min) (ng*hr/mL) % F^(‡)Study 1 15 mg Formulated 8 75.5 (48.7)  111 (38.3) 133 (72.0) 12.2(72.0) (SC424) 15 mg Unformulated 8 ND — — 0.0 Study 2 30 mgUnformulated 3 ND — — 0.0 (SC430) 30 mg Formulated*  4*  121 (35.5)*  95(36.0)*  367 (43.3)*  14.9 (43.3)* 45 mg Formulated 5 177 (62.5) 100(31.6) 574 (67.7) 15.5 (67.7) *Results reported excluding animal 5256^(†) Formulated capsules targeted 500 mg citric acid and 100 mg LLC^(‡)% F calculated based on that study's respective IV arm

Example 5: Fenofibrate Solubility Methods

Fenofibrate is a BCS class II compound and as such is insoluble inwater. It has solubility of 1 mg/mL in ethanol, 30 mg/mL in DMF, 15mg/mL in DMSO and 250 mcg/mL in 1:3 DMF:PBS pH 7.2

The solubility of fenofibrate in water was assessed at increasingconcentrations of LLC from 0.0% w/v to 10.0% w/v. Excess fenofibrate wasweighed into individual PP vials. Fenofibrate containing solutions weremixed at 125 rpm at 25° C. for 4 days. Dissolved fenofibrate wasmonitored by HPLC against a standard curve prepared in neat CH₃CN.

Results and Discussion

Data shown in FIG. 57 indicate increased solubility of fenofibrate inwater with increasing concentrations of LLC. The increased solubility offenofibrate may indicate the utility of LLC in enhancing solubility ofother class II molecules in water. LLC may be utilized in a compositionof the present disclosure as a solubility enhancer.

1. A solid oral dosage form comprising: a mixture comprising: atherapeutically effective amount of at least one active pharmaceuticalcompound classified as BCS Class III, wherein the compound does notinclude a peptide bond in the molecular structure of the compound; anabsorption enhancer; and coated organic acid particles, wherein theorganic acid particles are coated with a water soluble coat; an entericcoating; and a water soluble barrier positioned between the mixture andthe enteric coating, thereby separating the mixture from the entericcoating, wherein oral administration results in a synergistic increasein a systemic bioavailability of the active pharmaceutical compound whencompared to the systemic bioavailability provided by administration of asolid dosage form containing an equal dose of the active pharmaceuticalcompound without the absorption enhancer and the coated organic acidparticles.
 2. The solid oral dosage form of claim 1, wherein the organicacid is a carboxylic acid selected from the group consisting ofacetylsalicylic acid, acetic acid, ascorbic acid, citric acid, fumaricacid, glucuronic acid, glutaric acid, glyceric acid, glycocolic acid,glyoxic acid, isocitric acid, isovaleric acid, lactic acid, maleic acid,oxaloacetic acid, oxalosuccinic acid, propionic acid, pyruvic acid,succinic acid, tartaric acid, valeric acid and combinations thereof. 3.The solid oral dosage form of claim 2, wherein the coated organic acidis present in a quantity which, if the dosage form was added to tenmilliliters of 0.1 M aqueous sodium bicarbonate solution, the pH of thesolution would be acidified to a pH no higher than 5.5, wherein theorganic acid particles are coated with a water soluble coat thatseparates the organic acid from the active pharmaceutical compound. 4.The solid oral dosage form of claim 2, wherein the water soluble coatseparates the organic acid from the at least one active pharmaceuticalcompound.
 5. The solid oral dosage form of claim 2, comprising from 100mg to 500 mg of coated organic acid particles.
 6. The solid oral dosageform of claim 1, wherein the absorption enhancer is a combination of twoor more absorption enhancers.
 7. The solid oral dosage form of claim 1,wherein the absorption enhancer comprises a surface acting agent.
 8. Thesolid oral dosage form of claim 7, wherein the surface acting agent isan acid soluble bile acid.
 9. The solid oral dosage form of claim 1,wherein the absorption enhancer comprises an acylcarnitine.
 10. Thesolid oral dosage form of claim 1, wherein the absorption enhancercomprises lauroyl carnitine.
 11. The solid oral dosage form of claim 1,wherein the at least one active pharmaceutical compound classified asBCS Class III is an antibiotic or an antiviral compound.
 12. The solidoral dosage form of claim 1, wherein the at least one activepharmaceutical compound classified as BCS Class III is anaminoglycoside.
 13. The solid oral dosage form of claim 1, wherein theat least one active pharmaceutical compound is selected from the groupconsisting of tigecycline, zanamivir, kanamycin, and tobramycin.
 14. Asolid oral dosage form comprising: a mixture comprising: atherapeutically effective amount of at least one active pharmaceuticalcompound classified as BCS Class II, BCS Class III or BCS Class IV,wherein the compound does not include a peptide bond in the molecularstructure of the compound; an absorption enhancer; and coated organicacid particles, wherein the organic acid particles are coated with awater soluble coat; an enteric coating; and a water soluble barrierpositioned between the mixture and the enteric coating, therebyseparating the mixture from the enteric coating, wherein oraladministration results in a synergistic increase in a systemicbioavailability of the least one active pharmaceutical compound whencompared to the systemic bioavailability provided by administration of asolid dosage form containing an equal dose of the least one activepharmaceutical compound without the absorption enhancer and the coatedorganic acid particles.
 15. A method of treating a bacterial infectionor a viral infection in a subject in need thereof comprising orallyadministering a solid oral dosage form comprising: a mixture comprising:a therapeutically effective amount of at least one active pharmaceuticalcompound classified as BCS Class II, BCS Class III or BCS Class IV,wherein the compound does not include a peptide bond in the compound'smolecular structure; an absorption enhancer; and coated organic acidparticles, wherein the organic acid particles are coated with a watersoluble coat; an enteric coating; and a water soluble barrier positionedbetween the mixture and the enteric coating, thereby separating themixture from the enteric coating, wherein oral administration results ina synergistic increase in a systemic bioavailability of the least oneactive pharmaceutical compound when compared to the systemicbioavailability provided by administration of a solid dosage formcontaining an equal dose of the least one active pharmaceutical compoundwithout the absorption enhancer and the coated organic acid particles.